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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 University, 1969 M.D., Albany Medical College, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Experimental Pathology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1985 ®Donald Raymond Howard, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 8, 1985 DE-6(3/81) i i ABSTRACT D i f f e r e n t i a t i o n within the lymphoid system produces e f f e c t o r c e l l s which are involved i n a v a r i e t y of immune functions. For T c e l l s these include the provision of help, suppression, c y t o l y t i c a c t i v i t y and the regulation of cooperative c e l l u l a r i n t e r a c t i o n s . The primary function of B lineage c e l l s i s the production of s p e c i f i c antibody. Understanding the regulation of normal lymphocyte 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 may lead to a better appreciation of those factors 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 majority of which are B c e l l i n o r i g i n . Despite advances i n immunology and molecular biology, l i t t l e i s known about the mechanisms involved i n 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 or about those events leading to t h e i r malignant transformation. The advent of monoclonal antibody technology a decade ago has revolutionized our a b i l i t y to i d e n t i f y and characterize c e l l surface antigens. Because the a c t i v a t i o n and control of p r o l i f e r a t i o n of B c e l l s was already known to involve structures at the c e l l surface, i t was l o g i c a l to u t i l i z e monoclonal antibodies to i d e n t i f y a d d i t i o n a l c e l l surface molecules that might be important i n the function of normal B lymphocytes and that might allow normal and various types of neoplastic B c e l l s to be distinguished. To achieve t h i s goal, we developed monoclonal antibodies 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 large a c t i v e l y d i v i d i n g lymphoma c e l l s and small i n a c t i v e (quiescent) lymphocytes. These were tested for t h e i r a b i l i t y to i n h i b i t various T and B lymphocyte functions ( i . e . responses to anti-u, lipopolysaccharide, phytohemagglutinin and the mixed lymphocyte response) as i i i 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 cell 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 i i LIST OF TABLES vi LIST OF FIGURES v i i i ACKNOWLEDGEMENTS xi Chapter I THE LYMPHOID SYSTEM 1) General Concepts of Lymphoid Differentiation 1 A) The Cell Surface 1 B) Monoclonal Antibodies 3 C) Lymphocyte Ontogeny, Subpopulations and Differentiation Antigens 3 D) Lymphocyte Cell Surface Receptors and Gene Rearrangement 9 2) Mechanisms of B Cell Activation 11 A) Cell Surface Interactions 11 B) Growth Factors 15 3) Cell Surface Antigens on Normal and Neoplastic Human B-Lymphocytes 19 A) Definition Using Monoclonal Antibodies 19 B) The Human Major Histocompatibility Complex (HLA System) 23 C) Lymphocyte Function Associated Antigen (LFA) Family of Molecules 28 4) Neoplasms of the Immune System: The Non-Hodgkin's Lymphomas 31 5) Thesis Objectives 39 References 40 Chapter II MATERIALS AND METHODS 1) Cells 62 2) Monoclonal Antibodies 63 3) Preparation of Ascites 65 4) Purification of Antibody 66 5) Binding Assays 66 6) Antibody Coupling Procedure 67 7) Antibody Labeling 67 8) Stimulation Assays 68 9) Inhibition Assays 69 10) Colony Assays 70 11) Purification of B Cells 70 12) FACS Analysis 71 13) Immunoprecipitations 71 14) Antibody Blocking Studies 73 References 74 V Chapter III LYMPHOCYTE FUNCTION ASSOCIATED ANTIGEN (LFA-1) IS INVOLVED IN B CELL ACTIVATION 1) Introduction 76 2) Results 77 A) Monoclonal Antibody NB-107 Defines a Distinct Epitope on the LFA-1 Molecule 77 B) Expression of NB-107 on Peripheral Blood Mononuclear Cells, Neoplastic and Non-Neoplastic Cell Lines 79 C) NB-107 (Anti-LFA-1) Inhibits B Cell Activation 83 3) Discussion 89 References 96 Chapter IV MONOCLONAL ANTIBODIES TO HLA-CLASS II DETERMINANTS: FUNCTIONAL EFFECTS ON THE ACTIVATION AND PROLIFERATION OF NORMAL AND EBV TRANSFORMED B CELLS 1) Introduction 98 2) Results 99 A) Antibody Specificity 99 B) Inhibition of PBMC Stimulation 106 C) Inhibition of Purified B Cells 110 D) Inhibition of EBV Cell Lines 110 3) Discussion 110 References 118 Chapter V TWO MONOCLONAL ANTIBODIES THAT DEFINE UNIQUE ANTIGENIC DETERMINANTS ON B-LYMPHOMA CELLS 1) Introduction 120 2) Results 121 A) Reactivity with Cell Lines 121 B) Reactivity with Fresh Tissues 123 C) Reactivity with Normal B-Blasts 129 3) Discussion 129 References 136 Chapter VI SUMMARY AND CONCLUSIONS 139 vi LIST OF TABLES Page TABLE I Monoclonal antibodies defining B cell and B cell related surface determinants 21 TABLE II A comparison of the proposed "Working Formulation" with classifications for non-Hodgkin's lymphomas 36 3 TABLE III 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 cell line growth by anti-LFA-1. 90 TABLE IX Inhibition of anti-y stimulation: Purified B cells 91 TABLE X Effect of anti-LFA-1 on bone marrow progenitor cells 92 TABLE XI Reactivity of anti-class II monoclonal antibodies with homozygous DR cell lines: FACS analysis 101 125 TABLE XII Cross blocking of I-labeled anti-class II antibodies: DHL-4 cells 104 TABLE XIII Inhibition of stimulation of normal PBMC by anti-class II monoclonal antibodies 108 TABLE XIV Inhibition of anti-u stimulation of normal PBMC: Titration using purified anti-HLA class II antibody 109 TABLE XV Inhibition of mixed lymphocyte reaction by 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 Inhibition of EBV cell line proliferation by anti-class II monoclonal antibodies 114 TABLE XIX Cell line reactivity of antilymphoma antibodies: FACS analysis 122 v i i TABLE XX Analysis of fresh tissues: B cell malignancies -positive TABLE XXI Analysis of fresh tissues: B and T cell malignancies - negative TABLE XXII Analysis of fresh tissues: Reactive lymphoid proliferations - negative TABLE XXIII Analysis of fresh tissues: Miscellaneous - negative 124 127 128 130 v i i i LIST OF FIGURES 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 is illustrated in more detail in Figures 2 and 3. FIGURE 2 Model of T lymphocyte transformation. Resting T cells must first be activated before they are able to respond to interleukin-2 (IL-2). Page 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 first 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 cell 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 FIGURE 4 Follicular center cell 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 cell 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 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. 78 ix 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. 80 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. 82 FIGURE 8 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 FIGURE 9 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 is 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 FIGURE 10 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 1 2 SI-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 is 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. Sequential immunoprecipitation "preclearing" of 1 2^I-labeled WALK (DR4) cell lysate. "A" is 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 al l material reactive with itself, while not quantitatively affecting the amount of material precipitated by DH-84. 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. 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 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 cell based on size (e.g. large transformed lymphoid cells) within a given lymphoma. FACS histogram of purified LPS stimulated normal B cell 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 is only five per cent residual contamination with T cells (D). x i ACKNOWLEDGEMENTS "There i s a tide i n the a f f a i r s of men which, taken at the flood, leads on to fortune; omitted, a l l the voyage of th e i r l i f e i s bound i n shallows and i n miseries. On such a f u l l sea are we now a f l o a t , and we must take the current when i t serves, or lose our ventures." W. Shakespeare ( J u l i u s Caesar) I wish to express my gratitude To Drs. A. EAVES, C. EAVES and F. TAKEI for providing the opportunity, f a c i l i t i e s and stimulation necessary to complete t h i s work, To Drs. C. TAYLOR, J. BATSAKIS and B. MACPHERSON who, by t h e i r example, i n i t i a t e d me into the world of academic pathology, To C. SMITH, D. NIPIUS, W. DRAGOWSKA, and the other technologists from whom I have learned or who have assisted with this project, To J . WAITE and M. COULOMBE who have had the patience and s k i l l to type t h i s thesis, To T. FOX who inspired me, To A. LANCASTER who supported me, To DENISE who suffered with me, MARISSA and TREVOR who made me happy, and To M.V. NERY who started i t a l l . C H A P T E R I 1 THE LYMPHOID SYSTEM "There are two kinds of confidence which a reader may have i n his author... there i s a confidence i n facts and a confidence i n vision...The former requires simple f a i t h . The l a t t e r c a l l s upon you to judge for yourself and form your own conclusions." Anthony Trollope 1) GENERAL CONCEPTS OF LYMPHOCYTE DIFFERENTIATION (A) The C e l l Surface Many important p h y s i o l o g i c a l functions are mediated at the surfaces of c e l l s . These include: s e l e c t i v e transport of small molecules and ions, c e l l - c e l l 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 factors and mitogens, phagocytosis, exocytosis and endocytosis and metabolic regulation (1). The l i p i d b i l a y e r determines the basic structure of b i o l o g i c a l membranes. However, proteins are responsible for most membrane functions and serve as s p e c i f i c receptors, enzymes or transporters. The matrix of c e l l membranes i s a b i l a y e r composed predominantly of phospholipids, g l y c o l i p i d s and c h o l e s t e r o l . These molecules are amphipathic and associate with t h e i r more hydrophobic portions oriented i n t e r n a l l y and t h e i r h ydrophilic ends protruding externally. Embedded i n the b i l a y e r are the i n t e g r a l proteins of the membrane. Many of these are transmembrane proteins; most are glycoproteins. At physiological temperatures, the membrane e x i s t s as a two-dimensional f l u i d , i n which protein and l i p i d components unless s p e c i f i c a l l y r e s t r i c t e d , may move f r e e l y . The c e l l 2 membrane does not exist in isolation. 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 internal (cytoskeleton) environments (e.g. microfilaments, intermediate filaments and microtubules in the cytoskeleton; fibronectin and collagen in the exoskeleton (1, 2). Recent evidence has implicated a quantitatively minor group of membrane phospholipids (the polyphosphoinositides) in signal transmission for a wide variety of growth factors, neurotransmitters and hormones. Activation of the polyphosphoinositide system results in the release of products which act as "second messengers" in evoking the cell's responses. Two products released from polyphosphoinositide as a consequence of receptor activation are inositol triphosphate and diacylglycerol. Inositol triphosphate causes an increase in intracellular 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 of this system in lymphocyte activation i s just beginning to be explored. However, recent evidence suggests that perturbations of the T3-antigen receptor complex by monoclonal antibodies results in the release of inositol triphosphate. This in turn causes the release of calcium ions from intracellular stores; an effect which is thought to be important in the activation of the human T c e l l line, 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 inositol hydrolysis, followed by the generation of diacylglycerol and protein kinase C activation (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. First developed by Kohler and Milstein in 1975, monoclonal antibodies promise to be of major value in 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 characteristics of each. Usually, a mouse of the same genetic background as the myeloma cells is immunized with an antigen. The spleen of the mouse is removed and teased apart to yield a c e l l suspension. Myeloma cells and splenic B cells are then fused together to form a single c e l l or hybrid. Fusion is facilitated using polyethyleneglycol, an electrical pulse or Sendai virus. Hybrids are then selected, screened for antibody production, cloned, rescreened, recloned and fi n a l l y the antibodies are characterized. Inherited from the parent myeloma c e l l is the property of immortality in culture; from the immune lymphocyte the production of specific antibody. Monoclonal antibodies, because of their exquisite specificity can be used to detect distinct c e l l surface molecules or epitopes. Furthermore, monoclonal antibodies may be used to purify antigens or to assess biologic functions in in vitro assays (8). To a great degree, monoclonal antibodies have been responsible for the characterization and increased understanding of lymphocyte differentiation that has occurred in the last decade. (C) Lymphocyte Ontogeny, Subpopulations and Differentiation Antigens It is now clear that there are two classes of lymphocytes each mediating distinct functions (Figure 1). B cells secrete antibody; T cells subserve 4 Differentiation vs Transformation Hemopoiesis PHSC Lymphopoiesis Lymphoid Progenitor Cell Pre-B cell B-lmmunoblast*—mature B cell Pre-T cell mature T c e l l * * * T-lmmunoblast Plasma cell Memory B lymphocyte Effector Memory T lymphocyte T lymphocyte FIGURE 1 Schematic diagram of lymphocyte d i f f e r e n t i a t i o n -Beginning with the pluripotent hematopoietic stem c e l l (PHSC) lymphocytes pass through a serie s of stages ending with f u n c t i o n a l l y mature c e l l s of B or T lineage ( v e r t i c a l arrows). For contrast, the transformation of mature B and T lymphocytes to large a c t i v e l y d i v i d i n g immunoblasts i s represented by the horizontal arrows. Immunoblasts may d i f f e r e n t i a t e further to e f f e c t o r c e l l s of T or B lineage (e.g. cytotoxic T c e l l s , plasma c e l l s ) . The mechanism of transformation i s i l l u s t r a t e d i n more d e t a i l i n Figures 2 and 3. 5 regulatory and e f f e c t o r functions. In birds, B c e l l s are generated i n the lymphoid organ c a l l e d the bursa of Fa b r i c i u s . In mammals, hematopoietic stem c e l l s migrate from the yolk 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 into erythroid, myeloid and B c e l l s . Stem c e l l s then populate the bone marrow which becomes the major organ of hematopoiesis. Thereafter, B c e l l s and other c e l l s are continuously produced i n marrow throughout l i f e (9). B lymphocytes are categorized into functional subpopulations on the basis of the d i f f e r e n t classes of immunoglobulin they synthesize. B lymphocytes and t h e i r terminally d i f f e r e n t i a t e d progeny plasma c e l l s synthesize and/or secrete a l l classes of immunoglobulin molecules (IgM, IgG, IgA, IgD and IgE). Evidence now supports the hypothesis that the e a r l i e s t progenitors of ant i g e n - s p e c i f i c B c e l l s possess receptors of the IgM c l a s s . More mature B c e l l s express both c e l l surface IgM and IgD. Further maturation may r e s u l t i n the expression of IgG with or without IgD. So-c a l l e d "memory" B lymphocytes may express surface IgG, while " v i r g i n " B lymphocytes are thought to have IgM on th e i r c e l l surfaces. Memory B c e l l s are f u n c t i o n a l l y important for development of rapid secondary (anamnestic) antibody responses upon subsequent antigenic exposure (10). I t i s clear that B c e l l s undergo a process of immunoglobulin heavy chain switching during d i f f e r e n t i a t i o n , Isotype switching may be observed during c l o n a l p r o l i f e r a t i o n of B c e l l s i n response to c e r t a i n antigens or mitogens such as LPS. Constant heavy chain (CH) region isotype switching may also occur during the pre-B to B c e l l t r a n s i t i o n . At this stage of development the switching process i s most probably independent of antigen and T c e l l s . The function of these c e l l s , switched at early stages, i s unknown. Isotype switching may be either sequential, from IgM to other immunoglobulin isotypes i n order of the CH genes on the chromosome, or d i r e c t , from IgM to any of the 6 other isotypes encoded by downstream CH genes * Evidence suggests that the most commonly used pathways are direct 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 delineating different subpopulations of B c e l l s based on surface antigens, a functional correlation analogous to that i n the T lymphocyte system has yet to be shown. However, evidence does exist 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 their 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 signals delivered by B c e l l growth factor (BCGF) (19). B c e l l heterogeneity i s also evident from studies of their physical properties (e.g. s i z e , tissue d i s t r i b u t i o n and charge) and from their functional characteristics ( r e a c t i v i t y to different mitogens, antigens, genetic requirements for activation 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 induction). I t i s s t i l l not e n t i r e l y clear whether this heterogeneity r e f l e c t s changes associated with the clonal expansion of a single l i n e of B c e l l s or whether i t arises as a result of the generation of d i s t i n c t sublines of B c e l l s (20-22). Support for 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 deficiency determined by the X chromosomal gene Xid. 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 certainty 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 series 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 correlate with thymic d i f f e r e n t i a t i o n and the acquis i t i o n of T c e l l functions. Approximately 10% of the t o t a l human thymocyte pool display markers of early c o r t i c a l thymocytes: T i l , T10 and T9. 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 peripheral 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 into two d i s t i n c t lineages; one displaying 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 functionally mature and can be distinguished on the basis of T4 and T8 expression. T8+ c e l l s primarily mediate T c e l l c y t o t o x i c i t y . T4+ c e l l s act as effector c e l l s for delayed hypersensitivity, provide "help" for B c e l l and cyt 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 well defined. However, T4 and T8 appear to be involved respectively i n the recognition of MHC class I I and class I antigens. T3 i s closely associated with the T c e l l receptor. T9 i s i d e n t i c a l with the receptor for 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 activation structure through which the effects of phytohemagglutinin (PHA) are mediated (9, 24-29). Postulated c e l l u l a r interactions involved i n T c e l l activation 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, this 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-2R T-mitogens IL-2 Proliferation Differentiation Factors (?) Effector T cell FIGURE 2 Model of T lymphocyte transformation. Resting T c e l l s must f i r s t be activated before they are able 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 self-renewing cell population that contains the precursors of the peripheral T-cell 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 cell receptor for antigen has been characterized as a class of cell surface heterodimers, termed Ti, 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 cell receptors (Ig) and T cell receptors (Ti) have been cloned (34-36) and found to exhibit considerable homology (37). During the process of B and T cell differentiation Ig and Ti genes are rearranged. Rearrangement precedes the cell 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 cell leukemia. Moreover, immunoglobulin and T cell receptor gene rearrangement can be used as a diagnostic criterion for malignancies of B and T cell 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 cell (PHSC) to functionally mature B cell, 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 cell 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 its 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 cell). 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 cell 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 cell lineages. During the activation process both T cells and accessory cells produce lymphokines to which B lymphocytes respond. T cells produce B cell growth factor (BCGF), interleukin 2 (IL-2), B cell 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)+(^ ) Ag BCGF-R I Activated] BCGF B Proliferation BCGF-R T-'independent' mitogens BCDF TRF Plasma cell FIGURE 3 Simplified schematic diagram of B lymphocyte transformation. Figure shown i l l u s t r a t e s the c l a s s i c a l concepts of how normal B c e l l s transform into B immunoblasts i n response to antigen or mitogens. According to this model B c e l l s must f i r s t be activated by antigen or mitogen before being capable of responding to B c e l l 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 r e s t i n g B c e l l s to become more responsive to s t i m u l i such as anti-immunoglobulin. See text for d e t a i l s . 13 Step 2. The second step in B cell activation involves the transition of the B cell 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). Recent work, however, suggests this model may be oversimplified. It appears that antigen must first be internalized and processed by specific B cells before it 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 cell proliferation and maturation events. Various T cell and accessory cell 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 cell surface immunoglobulin (74). Lipopolysaccharide (LPS) likewise induces B cell 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 cell 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 cell 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 cell 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 cell 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). These factors are produced primarily by lymphocytes and monocytes. Those factors most relevant to B cell 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 cell 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 various inflammatory processes. IL-1 enhances the production of lymphokines by T lymphocytes. Its effects on B lymphocytes are less clear. However, it 16 appears that IL-1 can directly augment B lymphoproliferative and antibody-producing 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. IL-2 has been purified and its gene cloned. The molecular weight of IL-2 is approximately 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 it 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 in 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 cell 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 cell 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 cell 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 its gene cloned also (102). B Cell Stimulatory Factors B cell 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 its 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. This factor (human) has a molecular weight of approximately 12,000 by SDS-PAGE. When its purification is complete or its gene cloned, it 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 anti-immunoglobulin (e.g. anti-u) (104). B cell stimulatory factors have been derived from mitogen stimulated normal T cells, T hybridomas, T leukemia cell lines and HTLV-1 transformed T cell lines and are distinct from IL-2 (104-110). Most early studies supported the concept that anti-u induces cell enlargement, transition from GO to Gl of the cell cycle, and expression of receptors for BSF-pl. BSF-pl then induces entry of the cells into S phase (50, 52, 62-64, 74-78). 18 This concept has recently been challenged. Evidence has been provided which suggests that BSF-pl may be a differentiation or activation factor instead of, or in addition to, a growth factor. These studies have shown that BSF-pl acts upon resting B cells in the absence of anti-immunoglobulin (Ig) antibodies, to prepare these cells to respond to anti-Ig. Anti-Ig, possibly in conjunction with BSF-pl then induces entry of the cells into S phase (111-114). The differences between earlier and more recent studies apparently relate to methodological considerations. These include culture conditions, c e l l washing, and timing of the addition of the various factors and antisera to purified populations of B ce l l s . The complete significance of these findings remains to be elucidated. They may, however, account for the inability to clone and reproducibly propagate, normal factor dependent B cells in long term culture, as have been T cells (115-118). Recently, a monoclonal antibody to BSF-pl has been prepared. This antibody has allowed characterization of this B-cell factor and clearly shown that i t is distinct from IL-1, IL-2 and IL-3. It was recommended that the 'p' for provisional be removed and this lymphokine be henceforth referred to as BSF-1 (119). B Cell Differentiation Factors (BCDF) B c e l l differentiation factors act on post-activated B cells to induce differentiation to immunoglobulin secreting ce l l s . These factors have received a variety of names and may be a family of molecules rather than a single lymphokine. These include: B c e l l differentiation factor (BCDF), T c e l l replacing factor (TRF), B maturation factor (BMF) and B cell-derived enhancing factor (BEF) (106, 120-131). These factors, in 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 cell 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 cell 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 cell 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: 1. Diagnosis of immunodeficiency disorders. 2. Subtyping of acute lymphoblastic leukemia (ALL). 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 is 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 cell restricted (react only with cells of B lymphocyte lineage), B cell 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 cell lines (14-18, 152-170) (see Table I). The function of the antigens defined by these antibodies is 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 cell 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 LN-1, LN-2 BI, B2, B4, PC-1 Ia, CALLA, TI, T10 PCA-1, PCA-2 OKB1 0KB2 0KB4, OKB7 CB2 FMC7 HK-9, HK-19, HK-20 B220 ABl B cell subset B cell associated B cell restricted B cell associated B cell subset B cell associated B cell restricted SIg positive B cells B cell subset Mature B cells (DR related) B cells, some bone marrow cells Activated B cells (BSF receptor) McKearn et al, 1984 (14) Epstein et al, 1984 (15) Anderson et al, 1984 (16) Anderson et al, 1984 (152) Mittler et al, 1983 (17) Knowles et al, 1984 (18) Nadler et al, 1981 (157) Jephthah et al, 1984 (153) Zola et al, 1984 (154) Brooks et al, 1981 (155) Shipp et al, 1983 (156) Sarmiento et al, 1982 (170) Jung and Fu, 1984 (161) TABLE I (continued) Monoclonal Antibodies Defining B Cell and B Cell Related Cell Surface Determinants Antibody Specificity Reference Tac 33.1 BB-1 LB-1 4F2 5E9 B532 GB 1,2,3,5,6 8,10,11,13,14 38.13 41H.16 AB89 Activated B cells (IL-2 receptor) Activated B cells B cell blasts B and T cell blasts Activated lymphocytes, monocytes, embryonic fibroblasts Dividing cells (transferrin receptor) Activated B cells Some B cell lymphomas variable reactivity with B cells Burkitt lymphoma cells, some B lymphoma/leukemia cells some EBV transformed cell lines Normal and EBV transformed B cells, B-CLL B cell lymphomas (10%) Tsudo et al, 1982 (158) Marti et al, 1982 (159) Yokochi et al, 1982 (160) Kehrl et al, 1984 (163) Sutherland et al, 1981 (164) Frisman et al, 1983 (162) Funderud et al, 1983 (165) Klein et al, 1983 (166) Lipinski et al, 1982 (167) Zipf et al, 1983 (168) Nadler et al, 1980 (169) 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 it was observed that sera from multiply transfused patients and multiparous women contained antibodies reactive with leukocytes of non-identical donors. Since that time our appreciation of the complexity of the HLA system has increased enormously. The human HLA complex is located on the short arm of chromosome 6. 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~micro6l°bulin (encoded on human chromosome 15). 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 class II antigen system appears to be even more complex. Class II antigens are heterodimeric transmembrane glycoproteins composed of a heavy (a) chain of 33-35 Kd and a l i g h t (3) chain of 27-29 Kd. Class II antigens are found p r i n c i p a l l y on B lymphocytes, activated T lymphocytes and antigen 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 ) . They are also present on some myeloid c e l l s and thei r precursors. Low l e v e l s of cl a s s I I antigens have been reported on renal tubule c e l l s and on endothelial c e l l s . Class II antigens have also been reported on tumor c e l l s i n c l u d i n g melanomas and gliomas (174, 180, 181). Factors present i n mitogen activ a t e d T c e l l supernatant as well as pure y-interferon are capable of inducing c l a s s II antigen expression on class II negative macrophages, endothelial c e l l s and melanoma c e l l s (174, 182, 183). Biochemical and molecular genetic analyses of the genes and gene products associated with the HLA-D region indicate the existence of at lea s t three groups of products (180). The nomenclature for these products was recently revised (177). The designations now include HLA-DR, HLA-DQ (formerly DC, DS, MB) and HLA-DP (formerly SB). The genes coding for many of the class II products have been cloned and amino acid sequences of t h e i r protein products determined. A combination of serology, biochemistry and molecular biology indicates there are multiple class II genes coding for at least seven l i g h t (B) chains and at least s i x heavy (a) chains within the D region (184). Recent evidence supports the existence of at least one alpha and two or three beta chain genes within HLA-DR, two alpha and two beta chain genes within DQ, two alpha and two beta chain genes within DP, as well as an ad d i t i o n a l alpha chain which has previously been termed DZ-alpha (180, 185-188). It appears that the same heavy chain may associate with l i g h t chains from more than one locus and that determinants may be shared between 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'. This phenomenon is termed "linkage disequilibrium". 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). DP does not appear to be in linkage disequilibrium with DQ or DR (180). 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 (primed lymphocyte typing, PLT). Now, 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 cell 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. Most of these have been directed against monomorphic determinants. Some detect polymorphic class I and class II determinants (193-203). 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 it is preferable to refer to these as anti-class II monoclonal antibodies until it 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 cell lines or cell 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 cell 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 cell 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. These cells appear to be DR positive but DQ negative (217-220). The physiological significance of this latter observation is not yet clear. In summary, the MHC codes for two classes of cell 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 cell 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 11 and 6 precursors. 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 cell 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 variety 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: cytotoxic 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 allogeneic c e l l s , and antibody dependent c e l l u l a r cytotoxicity (ADCC). The role of LFA-1 i n B c e l l function has not been well characterized. 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 did 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 detailed studies on the role of LFA-1 i n immune function have centered on the relationship of this molecule to CTL mediated k i l l i n g . A n t i -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 participate 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 structure i s involved i n strengthening effector-target c e l l adhesion. Quantitative differences i n the a b i l i t y of different anti-LFA-1 monoclonal antibodies to block c y t o l y s i s indicate d i s t i n c t functional and antigenic epitopes exist on the LFA-1 molecule. With respect to CTL function anti-LFA-1 blocks k i l l i n g by binding to effector 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 related and suggests their 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 cell surfaces. Patients have severe impairment of adherence and adhesion dependent cell functions. Immune abnormalities described include: defective antibody dependent cellular cytotoxicity (ADCC), natural killer (NK) cell 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 clinical pathologic importance of lymphoid cell 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 glyco-protein. 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 cell 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 cell 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 cell" 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 config-uration 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 cell type (mixed lymphocytic-histiocytic lymphoma) and those that appeared especially primitive or "undifferentiated". Rappaport also demon-strated 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 histopath-ologic 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 undiffer-entiated 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 mito-gen. 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. Lymphoid neoplasms are characterized by the clonal proliferation of cells. In contrast, reactive cell 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 dissec-tion of the immune system into its functional components. Newer classifica-tions of the lymphomas have evolved in parallel with greater appreciation of the functional complexities of normal lymphocytes (252-261). These proposals remain hypotheses based on data indicating: (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 (268-274). 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 follicles (Figure 4). According to this new reasoning, cell size and nuclear configuration are not necessarily related to the degree of differen-tiation 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 may, by this concept, result in the morphologic expression of malignancy. 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 cell, B cell and true histiocytic (macrophage) types. A small proportion are undefined or unclassifiable by currently available techniques. The T and B cell 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 center c e l l concept of lymphocyte transformation. According to this hypothesis, normal B c e l l s pass through a series of morphologic stages within the f o l l i c u l a r centers of lymph nodes. B c e l l lymphomas may be c l a s s i f i e d according to which subtype of c e l l predominates. The predominant c e l l type, within a given lymphoma, may correspond to one of the stages i n normal B c e l l transformation i l l u s t r a t e d (252-254). TABLE II A Comparison of the Proposed "Working Formulation" with C l a s s i f i c a t i o n s f o r Non-Hodgkin's Lymphomas (274) WORKING FORMULATION Low Grade A. Small lymphocytic B. F o l l i c u l a r , small cleaved C. F o l l i c u l a r , mixed small cleaved and large c e l l Intermediate Grade D. F o l l i c u l a r , large c e l l E. Diffuse, small cleaved F. Diffuse, mixed, small and large c e l l G. Diffuse, large c e l l High Grade H. Large c e l l , immunoblastic I. Lymphoblastic (convoluted or nonconvoluted) J . Small noncleaved c e l l Others Hairy C e l l , Cutaneous T - C e l l , etc RAPPAPORT A. Well d i f f e r e n t i a t e d lymphocytic Poorly d i f f e r e n t i a t e d lymphocytic B. nodular E. d i f f u s e Mixed histiocytic-lymphocytic C. nodular F. d i f f u s e H i s t i o c y t i c D. nodular G. , H. d i f f u s e I. Lymphoblastic Undifferentiated J. Burkitt's J. pleomorphic LUKES-COLLINS ? U C e l l (Undefined) B C e l l A. Small Lymphocytic A. Plasmacytoid 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 ) (B or E) Small cleaved (D or G) Large cleaved (D or G) Large noncleaved (J) Small noncleaved H. Immunoblastic Sarcoma T C e l l A. Small lymphocytic I. Convoluted lymphocytic ? Cerebriform (cutaneous) F.G. Lymphoepithelioid c e l l H. Immunoblastic sarcoma H i s t i o c y t i c 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 it 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 cell lymphomas and leukemias. Some of these show a tendency to associate with specific histologic subtypes (289-292). However, these associations are not invariant. 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 first postulated by Todaro (294). More recently, B cell growth factors and differentiation factors have been reported to be products of neoplastic and EBV transformed cell lines (122, 132-136). Whether autocrine secretion of growth factors is important in the pathogenesis of B cell malignancy is not yet known. The putative causative agent of some human T cell 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, clinically 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 viral interactions occur at the cell surface, identification and characterization of molecules characteristic of or unique to lymphoma cells is of obvious biologic interest. 3 9 5) THESIS OBJECTIVES The c e l l surface is involved in many events which are crucial to the function of normal B lymphocytes. These include: c e l l activation and proliferation, c e l l - c e l l interactions, growth factor-receptor binding and the regulation of metabolic processes. A role for c e l l surface molecules in 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 specificity. The purpose of this research is 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 . 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C H A P T E R II 62 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 cells were obtained from peripheral blood from 2 normal volunteers after separation on 1.077 gm/cm Ficoll-Hypaque (LSM, Litton Bionetics, Bethesda, MD). Cells were obtained from tissues (lymph nodes, spleens etc) by gently teasing these apart, using forceps, into RPMI 1640 + 10% FCS under aseptic conditions. The larger fragments were then gently pressed through a fine wire mesh to further release cells and remove larger pieces of tissue. Bone marrow specimens were obtained in Heparin and the mononuclear fraction separated as above on Ficoll-Hypaque. Tissues and bone marrows were obtained from patients as part of their routine diagnostic evaluation. Cells were then washed in RPMI 1640 vith 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 in Falcon flasks at 10 6 cells/ml in RPMI 10% FCS vith 100 ug/ml of lipopolysaccharide (LPS, E. c o l i , Sigma, St. Louis, M0) for four days. T cell s vere depleted by E-rosette sedimentation using aminoisothiouronium bromide (AET) treated sheep red blood cells (SRBC) (1). The remaining cells vere 90% surface immunoglobulin positive B cells and vere large in size as 63 judged by forward scatter on the FACS. Morphologic examination of stained cytospins showed that these cells were predominantly large lymphoblasts. Cells prepared in this way were < 5% T cells and monocytes as shown by staining with Leu-5 and Leu-M3 (Becton Dickinson, Mountain View, CA). 9 EBV c e l l lines were the generous gi f t s of Drs. John Hansen and Paolo Antonelli (Genetic Systems, Seattle, VA) and the 8 lymphoma lines the kind g i f t s of Drs. Alan Epstein (Univ. S. California, 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 lines were maintained in RPMI 1640 medium plus 10-15% FCS and subcultured twice weekly or as required. 2) MONOCLONAL ANTIBODIES (C3HXBALB/c) Fl (bred in our colony) or BALB/c (Charles River Laboratories) mice were immunized with DHL-10 cells (DH-84, DH-224), DHL-10 membranes (LM-26, LM-155) or LPS stimulated spleen cells from a patient with B c e l l lymphoma and splenic involvement (NB-29, NB-65). Cells or membranes (10 7 c e l l s or equivalent membranes) were homogenized in Freund's complete adjuvant (Difco Labs, Detroit, MI) and injected intraperitoneally (IP) into mice. Two IP injections were given at least 3-4 weeks apart. Cells (2 x 10 7) or equivalent membranes in saline were administered intravenously (IV) 3-4 weeks after the last IP injection. Cell fusion took place the fourth day after IV boost according to established procedures (6). 9 Ce l l membranes were prepared by washing 2 x 10 DHL-10 cells with phosphate buffered saline (PBS) then resuspending these cells in 10 mM Tris HCL pH 8.0 plus PMSF (phenylmethylsulfonyl fluoride, Sigma, St. Louis, M0) (7). Cells were homogenized vith 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 in dH^ O and spun at 25,000 RPM for 60 minutes. Membranes were removed from the interface and washed with Tris HCL. The protein content of the pellet was measured at 280 nm, and the crude membrane material aliquoted and frozen at -70°C unt i l used. Prior to fusion, NS-1 cells were grown in Dulbecco's minimal essential medium (DMM) plus 5% fetal calf serum (PCS) in spinner flasks. Cells were washed 3 times in DMM immediately prior to fusion. The spleen was removed aseptically from an immune mouse. Cells were gently teased into DMM, f i l t e r e d to remove large clumps and washed. Spleen cells and NS-1 c e l l s were mixed at a ratio of 10:1, diluted to 50 ml in DMM and spun. After removing the supernate, 1 ml of 50% polyethylene glycol 1500 (PEG)/DMM was added slowly over one minute with constant agitation. Nine ml of DMM was then added slowly over the next five minutes. Fused cells were spun at 1,200 RPM for 5 minutes and resuspended slowly in 5 ml DMM + 15% FCS. Cells were then resuspended in 100 ml DMM + 15% FCS and 1 ml aliquoted into each well of plastic multiwell (24) plates (Flow Labs, McLean, VA). One ml of normal syngeneic spleen cells used as feeders were dispensed into each well at a concentration of 2 x 10^ cells/ml. Cells were cultured in a 37°C incubator at 5% CO2 and 100% humidity. On day one post fusion approximately 1 ml of supernate was removed from each well and replaced with hypoxanthine-aminopterin-thymidine (HAT) media. This procedure was repeated on days 3 and 6. On day 8 or 9 post fusion c e l l s were switched by a similar 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 dissolving 0.65 g hypoxanthine, 0.0095 g aminopterin and 0.195 g thymidine in 500 ml of 0.01 N NaOH dH20. Stock H-T was prepared by dissolving 0.135 g hypoxanthine and 0.039 g thymidine in 100 ml of NaOH.v Stock solutions were diluted 1:100 with 65 media prior to use. Final concentrations in culture of reagents vere hypoxanthine (13 ug/ml), aminopterin (0.19 jig/ml) and thymidine (3.9 pg/ml). Wells vere observed daily for hybridoma grovth. When hybrids appeared v e i l established, supernatants vere screened for antibody activity using the binding assay described belov. Antibody positive cups vere transferred to DMM 15% FCS in individual flasks and cups and rescreened prior to cloning. Hybridomas vere plated in methylcellulose media (40 ml 2% methylcellulose in alpha media + 1 ml glutamine + 1 ml 2 mercaptoethanol + 10 ml FCS + 30 ml DMM). Visible colonies vere detected in approximately one veek, plucked and transferred to individual multivell plates. When grovn, cultures vere rescreened for antibody activity and the cloning process repeated. Hybrids vere grovn in DMM gradually reducing the concentration of FCS to 5%. Cells vere frozen at appropriate stages of the cloning process and after double cloning, thaved and v i a b i l i t y checked. Antibody subclasses vere determined by Ouchterlony immunodiffusion in 1.2% agar using goat anti-mouse IgG subclass specific antisera (Tago Inc., Burlingame, CA). Antibodies vhich preferentially bound to large B-lymphoma cells (DHL-10) compared to small B-lymphocytes (CLL cells) by binding assay or FACS analysis (see belov) vere selected for further characterization. 3) PREPARATION OF ASCITES Hybrid clones vere thaved and grovn in DMM + 5% FCS. Cells vere vashed four times vith normal saline, resuspended in saline and then injected IP into pristane (2, 6, 10, 14-Tetramethylpentadecane, Aldrich Chem. Co., Milvaukee, Wl) primed mice (2 x 10 7 cells per mouse). Mice vere primed vith 0.5 cc of pristane IP 1 veek prior to being injected vith hybridomas. After 1 to 2 veeks ascites vere collected, spun at 2,000 RPM for 10 minutes and stored at -20°C. 6 6 4) PURIFICATION OF ANTIBODY Equal volumes of immune ascites and saturated ammonium sulfate were mixed and stirred for one hour at room temperature. The mixture vas spun at 10,000 RPM for 15 minutes and the supernate removed. The precipitate was then dissolved in 20 mM phosphate buffer pH 8.0 and dialyzed overnight against the same buffer. A DEAE Affi-Gel Blue column (Bio-Rad, Richmond, CA) vas prepared and washed sequentially with 0.5 M phosphate buffer pH 8.0 and 20 mM phosphate buffer pH 8.0. Dialyzed antibody vas loaded onto the column followed by 20 mM phosphate buffer. Samples vere collected in a fraction collector and each fraction individually analyzed for protein concentration at 0D 280 nm, binding activity to appropriate c e l l lines, and SDS-PAGE gel electrophoresis. Aliquots containing antibody vere pooled and frozen at -20°C. Preparation of rabbit anti-mouse immunoglobulin F(ab') 2 (RaMIg) was by a f f i n i t y purification using mouse immunoglobulin conjugated to sepharose beads (8). 5) BINDING ASSAYS Target cells (e.g. DHL-10, CLL) were washed in Earl's balanced salt solution (EBSS) + 0.5% BSA + 10 mM HEPES + 0.1% azide (binding assay media, BAM). Cells were resuspended at 107 per ml in 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 in duplicate. After a 1 hour incubation at 4°C, cells were washed twice in BAM. 10^ cpm of 125 I labeled F(ab')2 rabbit anti-mouse immunoglobulin in 50 ul of BAM was added to each well. After a one hour incubation at 4°C, cells vere vashed three times and resuspended in 100 ul 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 relative 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. Purified 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 in binding assay media and stored at 4°C. 7) ANTIBODY LABELING 125 I labeling was performed according to established procedures (9). A p30 column was prepared by placing a small piece of glass wool in a pasteur pipette which was then f i l l e d with a p30 sizing gel (Bio-Rad, Richmond, VA). The gel was washed with BAM to saturate protein binding sites, followed by phosphate buffered saline (PBS). 25-50 ug of antibody in 25-50 u l was added to 10 ul of chloramine T solution (0.5 mg/ml in dB^O, Sigma) in 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 ul of sodium bisulphite (20 pg/ml in PBS, Sigma) was added to stop the reaction. The mixture was added to the column of p30 followed by PBS. Fractions were collected in tubes and a gross estimate of the radioactivity made using a Geiger counter. Fractions containing labeled antibody were pooled diluted with BAM and stored at 4°C. 68 3 6 H-lysine labeling: 5 x 10 hybridoma cells vere vashed in lysine free DMEM media containing glutamine + 10% dialyzed FCS. Cells vere resuspended 3 in 5 ml of the same media containing 1 mCi H-lysine (Nev England Nuclear, Boston, MA) 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. Cells vere removed by centrifugation. To the supernatant an equal volume of saturated ammonium sulfate vas added, the mixture microfuged and the precipitate dissolved in saline. This procedure vas repeated 3 times. Finally, the 3 precipitate vas redissolved in BAM and dialyzed to remove unbound H-lysine. 8) STIMULATION ASSAYS Mitogen stimulation assays vere performed by modifying existing procedures (10, 11). Cells vere separated as above and resuspended in RPMI 1640 + 2 mM glutamine + Na pyruvate (110 mg/liter) + 10 mM HEPES buffer + 5% FCS at a concentration of 2 x 10** cells/ml. 0.1 ml (2 x 10 5 cells) vere placed in veils of flat 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 diluted in 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. Control veils contained ce l l s vithout mitogen in the same volume. Cultures vere incubated for 3 days (PHA) or 4 days (LPS, anti-p) at 37°C, 5% C0 2 and 100% humidity. One luCi of 3 H-thymidine (Amersham Int. Ltd., Amersham, UK, specific activity 2.0 Ci/mM) vas added to each v e i l for 4 hours. Cells vere harvested onto glass f i l t e r disks using a multiple automated sample harvester, dried, dissolved in s c i n t i l l a t i o n f l u i d and counted on a Beckman liquid s c i n t i l l a t i o n counter. 6 9 Stimulator cells for one-way mixed lymphocyte cultures (MLC) vere prepared by suspending PBMC at a concentration of 10 7 cells/ml in the above media and adding 0.1 ml stock, mitomycin C (Sigma, St. Louis, MO) per ml of c e l l suspension. Cells vere incubated 1 hour at 37°C and vashed 3 times vit h 5 5 media. 1.5 x 10 responder cells (untreated, vashed PBMC) and 1.5 x 10 mitomycin treated stimulator cells vere added to each v e i l of a microtiter plate in tr i p l i c a t e folloved by 50 ul of antibody or media as appropriate. 3 Cells vere cultured 4 days, pulsed vith 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 cells in media or cel l s in media plus mitogen. To each of these vas added 50 p i of hybridoma supernate or purified antibody. Purified antibodies vere titrated for their a b i l i t y to inhibit stimulation over a concentration ranging from 0.3 ug/ml to 50 pg/ml. A l l tests vere set up in triplicate and included negative controls (cells + media, cells + test monoclonal antibody), positive controls (cells + mitogen) and test samples (cells + mitogen) and test samples (cells + mitogen + antibody). Results are expressed as mean counts per minute (cpm) vith standard error of the mean (SEM) of tri p l i c a t e v e i l s . Percent inhibition vas calculated by dividing mean test cpm by positive control cpm after subtracting background counts according to the folloving equation: fl- (Test) - (antibody control) j \ (Positive control) - (Negative control)/ X 100. Those instances in vhich the numerator of this equation exceeds the denominator are reported as 0 percent inhibition. 70 Cell lines used for inhibition assays were cultured at an i n i t i a l density of 2 x 10 cells per ml in RPMI 1640 + 10% FCS either with or without 50 ul of antibody supernatant or 50 p i of purified antibody at various concentrations (1 to 20 jug/ml f i n a l concentration). Cells were pulsed with t r i t i a t e d thymidine, harvested on day three and counted as above. 10) COLONY ASSAYS Erythropoietic (CFU-E and BFU-E), granulopoietic (CFU-GM), and pluripotent (CFU-G/E) progenitors were assayed in 0.8% methylcellulose in Iscove's medium, supplemented with 30% FCS, 1% deionized BSA, 10"^ M 2-mercaptoethanol, 200 mM L-glutamine, 3 units/ml of human urinary erythropoietin (purified to a specific activity of > 100 u/mg) (12) and agar-stimulated human leucocyte conditioned medium (13) with or without the addition of purified NB-107 at a fi n a l concentration of 5 pg/ml. Histologically normal fresh marrow cells from two different donors were 5 assayed by plating 2 x 10 washed buffy coat cells per 1.1 ml of culture and mature erythroid, granulocyte-macrophage, and mixed colonies identified 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 over Ficoll-Hypaque. T c e l l depletion was then accomplished by incubation of these c e l l s with aminoethylisothiouronium 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 petri dishes overnight at 37°C. Using these two procedures B cells were enriched to 60-70% as determined by positivity for surface immunoglobulin. Partially purified B cells 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 c e l l sorter (FACS 440, Becton Dickinson, Sunnyvale, CA) were set to exclude T cells (fluorescein positive) and monocytes (on the basis of light scatter). The f i n a l B c e l l enrichment resulted in 85-90% B cell 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. Purified B cells were then set up in culture as described above. 12) FACS ANALYSIS Cells (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 in 50 (ul of undiluted culture supernatant or purified antibody. After a 1 hour incubation at 4°C, the cells were washed twice and resuspended in 50 ul of pretitrated FITC conjugated goat anti-mouse IgG F(ab')2 (Tago, Burlingame, CA). After an additional incubation at 4°C for 1 hour, cells were washed three times with PBS containing azide, fixed in 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 vith each experimental run. These included antibodies of irrelevant s p e c i f i c i t y (e.g. anti-Thy 1.2) as negative controls, and antibodies knovn to react vith hematopoietic c e l l populations of interest (e.g. anti-leukocyte, Becton Dickinson, Mountain Viev, CA) as positive controls. 13) IMMUN0PRECIPITATI0NS At least 2 X 107 viable DHL-4, DHL-10 or WALK cells vere vashed 3 times vith PBS. The cells vere counted, resuspended in 0.5 ml PBS and transferred 72 125 to an iodogen (Pierce, Rockford, IL) v i a l . 10 pCi of I were added and the mixture incubated on a rocker at room temperature for 1 hour with constant shaking. Cells were then washed 3 times with PBS, once with BAM and resuspended in 1.5 ml of 50 mM Tris-saline containing 0.5% BSA. 0.5 ml of 2% NP40 in 50 mM Tris-saline was added and the cells 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 ul counted in a gamma counter. A minimum of 2 x 10^ counts per tube of labeled c e l l lysate was u t i l i z e d . 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 Tris-saline buffer containing 0.5% BSA and 0.5% NP40. 40 pi of 50% suspension of washed beads was 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 Tris-saline and 150 p i of non-reducing sample buffer added to each tube. Samples were boiled 5 minutes and the supernate removed. Half of each supernate was 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 gel electrophoresis. Sequential immunoprecipitations were performed with cells 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, anti-transferrin 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 lasting overnight. Precleared supernates were then processed as above for ordinary immunoprecipitations. In some instances (the anti-class II antibodies) 73 complete removal of immunoreactive material could not be accomplished using this indirect technique. Therefore, these antibodies were purified, conjugated 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 after centrifugation. This process was repeated up to 5 times, with the last incubation lasting overnight. Following this direct preclearing, immunoprecipitation was performed in the usual way as described above. Reduced and nonreduced samples were run in a 10% polyacrylamide gel using a slab gel electrophoresis apparatus (Bio-Rad, Richmond, CA). 14) ANTIBODY BLOCKING STUDIES 125 3 Antibodies were labeled as above with I or H-lysine. Labeled antibodies were titrated and saturating levels 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 titrated and each shown to have significant reactivity with DHL-4, DHL-10 or WALK cells at titers in excess of 1:30,000. 10 cells per well (e.g. DHL-4) were placed into round bottom microtiter wells. Labeled antibody was mixed with an equal volume of s e r i a l l y diluted ascites. 50 ^ul of labeled-unlabeled antibody mixture was added to each well and incubated 1 hour at 4°C. Cells were washed 3 times with binding assay media, then counted in a Beckman liquid s c i n t i l l a t i o n counter. Controls for non-specific inhibition of binding included ascites f l u i d containing antibody against unrelated determinants present on test c e l l s . The percentage inhibition was calculated as a ratio of counts per well, containing cold antibody, to control wells without cold antibody. 74 REFERENCES 1. Madsen H , Johnsen H, Hansen P, Christiansen S: Isolation of human T and B lymphocytes by E-rosette gradient centrifugation. Characterization of the isolated subpopulations. J Immunol Meth 33: 323-336, 1980. 2. Nilsson K, Sundstrom C: Establishment and characteristics of two unique c e l l lines from patients with lymphosarcoma. Int J Cancer 13: 808-823, 1974. 3. Winter J, Variakojis D, Epstein A: Phenotypic analysis of established diffuse histiocytic lymphoma c e l l lines 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 nutritional requirements for the establishment and cloning of human malignant lymphoma c e l l lines. 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 diffuse histiocytic lymphoma c e l l lines. Cancer 42: 2379-2385, 1978. 6. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. 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 Edition, Weir DM (ed), Blackwell Scientific Publications, Oxford, Chapter 6, pp 6.1-6.102, 1978. 9. Markwell MAK, Fox CF: Surface specific iodination of membrane proteins of viruses and eukaryotic cells using l,3,4,6-tetrachloro-3a, 6a-diphenylglycouril. 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 in vitro. Eur J Immunol 4: 193-199, 1974. 11. Miller R, Gartner S, Kaplan H: Stimulation of mitogenic responses in human peripheral blood lymphocytes by lipopolysaccharide: Serum and T helper c e l l requirements. J Immunol 121: 2160-2164, 1978. 12. Krystal G, Eaves CJ, Eaves AC: CM Affi-Gel Blue chromatography of human urine: a simple one-step procedure for obtaining erythropoietin suitable ^ o r in vitro erythropoietic progenitor assays. Br J Haematol 58: 533-546, 1984. 13. Eaves CJ, Eaves AC: Erythropoietin (Ep) dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythemia vera. Blood 52: 1196-1210, 1978. 75 14. Askew DS, Eaves AC, Takei P: NHL-30.5: A monoclonal antibody reactive 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 activation. In: Leukocyte Typing II, 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, Milstein C: Monoclonal xenogeneic antibodies to murine c e l l surface antigens: identification of novel leukocyte differentiation 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 insignificant, but i t is very important that you do i t . " Gandhi 1) INTRODUCTION An interrelated family of three different c e l l surface molecules, expressed on hematopoietic cells of diverse types, has recently been identified. These molecules, termed LFA-1, Mac-1 (0KM1, Mo-1), and pl50,95, are defined by different monoclonal antibodies (1-3). Each possess a common, apparently identical, 6 subunit of Mr 95,000 and variable a subunits of approximately Mr 177,000, 165,000 and 150,000 respectively. The antigen defined by 0KM1 appears to be the complement receptor type three (CR3) (1). The specific functions of LFA-1 and pl50,95 are unknown. LFA-1 is a widely expressed human leukocyte antigen present on lymphocytes, monocytes, thymocytes, granulocytes and some bone marrow cells (2). LFA-1 has been shown to play an important role in a variety of T c e l l interactions. Monoclonal antibodies to LFA-1 have been shown to inhibit various T c e l l functions including: antigen-specific cytotoxic T lymphocyte (CTL) mediated k i l l i n g , natural k i l l e r (NK) cytolysis, T c e l l proliferative responses to antigen, mitogen and allogeneic cel l s , and T c e l l dependent plaque-forming c e l l responses (1-6). The mechanism of action of monoclonal anti-LFA-1 in 77 inhibiting the diverse cellular immune processes is largely unexplored, although evidence that anti-LFA-1 monoclonal antibodies block. CTL mediated k i l l i n g by inhibiting adhesion between the CTL and the target c e l l has been +2 reported. According to these studies LFA-1 may participate in the Mg -dependent adhesion step of CTL mediated k i l l i n g (2). Despite the demonstrated widespread importance of LFA-1 in T c e l l responses a role for LFA-1 in B c e l l function has not been documented. B cells may be induced to proliferate in response to a variety of stimuli including specific antigens as well as polyclonal mitogens such as lipopolysaccharide (LPS) and anti-IgM (|u) antibodies. The molecular interactions underlying B c e l l activation are largely unknown. At high concentrations, anti-u is thought to in i t i a t e a direct proliferative effect on B cells independent of T cell s , monocytes or their products ( 7 , 8 ) . In this chapter the inhibition of normal B c e l l activation by a monoclonal antibody to LFA-1 is reported, and evidence provided that this effect is mediated via action of the antibody on accessory cells or T lymphocytes. These findings document a previously unrecognized role for LFA-1 in the regulation of B c e l l proliferation and suggest a more generalized role for LFA-1 in 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 is 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 cells (Fig. 5). The molecules immunoprecipitated by NB-107 appear identical 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 preferentially with the B and a chains respectively of the LFA-1 molecule (1). Removal of material in DHL-4 c e l l lysates reactive with NB-107 by preclearing with this antibody, completely eliminated lysate reactivity with TS1/18 and TS1/22 indicating these antibodies react with identical molecules. In contrast, binding of antibody NB-65 to the transferrin receptor vas not significantly decreased by preclearing vith NB-107. Preclearing vith the same antibody to the transferrin receptor, hovever, did remove a l l detectable transferrin-receptor and did not affect immunoprecipitation using the anti-LFA-1 monoclonal antibodies (Fig. 6). That NB-107 reacts vith a distinct epitope on the LFA-1 molecule vas demonstrated by the antibody inhibition studies shovn in Table III. The binding of radioactively labeled NB-107 to B lymphoma cells vas nearly completely inhibited by cold purified NB-107 even at very high dilutions of cold antibody. In contrast, no significant inhibition 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 vith an epitope on the LFA-1 molecule that i s distinct from those defined by TS1/18 and TSl/22. (B) Expression of NB-107 on Peripheral Blood Mononuclear Cells, Neoplastic and Non-Neoplastic Cell Lines FACS analysis of NB-107 binding to peripheral blood mononuclear ce l l s from 27 normal individuals revealed a mean + SEM reactivity of 83 + 13 percent. Mean reactivity vith bone marrov cells from 7 patients (3 CML, 1 AML, 1 myelodysplasia, 1 monocytosis, 1 hairy c e l l leukemia) was 45 percent (range 21-72 percent). Figure 7 shows a FACS analysis of NB-107 tested against normal PBMC. Cells with the greatest amount of light 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 III 3 Competitive Inhibition of H-lysine Labeled NB-107 Binding to DHL-4 Cells Antibody Ascites Dilution NB-107 Binding (CPM) % Inhibition NB-107 200 400 800 1600 3200 201 240 372 621 1003 94 93 90 87 72 TS1-18 200 400 800 1600 3200 3208 3583 3692 3549 3500 9 0 0 0 1 TS1-22 200 400 800 1600 3200 3529 3616 3681 3706 3171 0 0 0 0 10 Control 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. Cells of intermediate size (lymphocytes) showed a spectrum of reactivity from strong to weak. Dual labeling of PBMC with phycoerythrin anti-DR and fluoresceinated NB-107 revealed that a major fraction of both DR positive c e l l s (B cells and monocytes) and DR negative cells (principally T cells) were positive for NB-107 (Fig. 8). Whether there is any functional significance 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 lines. 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 lines examined and 3 of 7 B lymphoma c e l l lines. Noteworthy was i t s absence on the other 4 B lymphoma c e l l lines tested. (C) NB-107 (Anti-LFA-1) Inhibits B Cell Activation NB-107 profoundly inhibited both LPS and anti-p induced proliferative responses of PBMC (Table V). This was true for both hybridoma supernate and purified antibody. Significant inhibition was observed over a wide range of antibody concentrations (Table VI). In a l l experiments isotype identical negative controls were performed and showed no significant inhibition. Antibody to transferrin receptor, a known inhibitor of c e l l proliferation served as a positive control (10). NB-107 inhibited the MLR, as previously reported for anti-LFA-1 monoclonal antibodies (11). In contrast to other studies, we did not observe inhibition 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 ce l l s , we attempted to inhibit the growth of EBV transformed B c e l l lines by addition of purified NB-107 to cultures of these cel l s . Two different EBV c e l l lines were tested and in neither case was evidence of significant 8 4 NB-107 FLUORESCEIN FIGURE 8 Dual fluorescence of normal peripheral blood mononuclear c e l l s using phycoerythrin labeled anti-DR and f l u o r e s c e i n labeled NB-107. C e l l s with the greatest i n t e n s i t y of DR st a i n i n g ( p r i n c i p a l l y monocytes) also express the highest amounts of LFA-1 defined by NB-107. TABLE IV Cell Line Reactivity of NB-107 (FACS Analysis) 85 Cell Line Type R e a c t i v i t y JREE CMG RMG WAIK SWEI ELD BN MAD KOZ EBV (DR1) EBV (DR2) EBV (DR3) EBV (DRA) EBV (DR5) EBV (DR6) EBV (DR7) EBV (DRW8) EBV (DRW9) SU-DHL-1 SU-DHL-4 SU-DHL-6 SU-DHL-8 SU-DHL-10 BALM-5 U-698-M B lymphoma B lymphoma B lymphoma B lymphoma B lymphoma B lymphoma B lymphoma Jurkat T leukemia HL-60 K562 Myeloid leukemia Erythroleukemia 86 TABLE V Inh i b i t i o n of B C e l l Activation by An t i - L F A - l a ^H-Thymidine Incorporation A »-w A (Mean CPM + SEM) „ T . Mitogen Antibody - ' % I n h i b i t i o n C o n t r o l 0 Test c A n t i - u d None 1167 + 192 3248 + 402 NB-65e 586 + 92 1735 + 129 69 + 21 NB-107 566 + 173 1456 + 119 71 + 21 LPS None 1946 + 752 4028 + 426 NHL 30.5 f 1338 + 190 3150 + 475 12 + 7 NB-65 491 + 21 901 + 125 68 + 16 NB-107 725 + 320 1349 + 83 80 + 14 aResults 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 wells containing 2 x 10^ peripheral 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 s i x i n d i v i d u a l experiments. DCultures without mitogens. cCultures with mitogens. ^ A f f i n i t y p u r i f i e d polyclonal goat anti-human IgM. eAntibody to tran s f e r r i n receptor used as a positive control. ^NHL 30.5 i s a subclass i d e n t i c a l monoclonal antibody directed 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 controls. 87 TABLE VI I n h i b i t i o n of LPS Stimulation: T i t r a t i o n Using P u r i f i e d NB-107 3 H-Thymidine Incorporation Antibody 3 ( M e a n C P M ± S E M ) % I n h i b i t i o n <r l8 / m l> C o n t r o l Test C 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 See Table V for description of antibodies. 'Without mitogen. With mitogen. 88 TABLE VII Inhibition of T Cell Proliferation Mean + SEM Antibody a Control Test 0 X Inhibition' MLR None 459 + 204 17,998 + 797 NHL 30.5 679 + 141 19,810 + 1941 0 NB-65 343 + 93 4,028 + 502 83 + 6 NB-107 219 + 104 3,811 + 826 85 + 7 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 for description of antibodies. Culture without stimulator cells or mitogen. Cultures with stimulator cells or mitogen. Wan + SEM of two individual experiments. 89 inhibition obtained (Table VIII). This was in contrast to the marked inhibition achieved by the addition of anti-transferrin receptor antibody. The proliferative response of highly purified normal B cells to anti-^u was, likewise, not inhibited by NB-107 (Table IX) nor did the presence of NB-107 have any inhibitory effect on colony formation by bone marrow CFU-E, BFU-E, CFU-GM or CFU-G/E assayed in standard methyl cellulose cultures (Table X). 3) DISCUSSION We have demonstrated that LFA-1 may be important in the regulation of B c e l l proliferation. Monoclonal antibody to LFA-1 (NB-107) profoundly inhibited stimulation of human peripheral blood mononuclear cells (PBMC) by LPS and anti-p. In order to further investigate whether NB-107 inhibits B lymphocyte mitogen induced activation via a direct action on B cells or by inhibiting an essential accessory c e l l function or T-B c e l l interaction, the effect of NB-107 on activation of highly purified B cells was tested. NB-107 antibody did not inhibit the anti-p stimulation of the B enriched populations, which contained less than 1% T cells (0KT4+), approximately 5% monocytes (0KM1+) and 85-90% surface immunoglobulin positive B ce l l s . 3 Furthermore, NB-107 also did not inhibit H-thymidine uptake of several EBV transformed B c e l l lines. Therefore, anti-LFA-1 does not seem to inhibit B cel l s directly. These findings raise the question of how the B c e l l response to anti-p involves other cells (such as T cells or monocytes) and how anti-LFA-1 inhibits the B c e l l response. Because anti-p stimulated highly enriched populations of B ce l l s , either this stimulation does not require T cells and/or monocytes, or those cells s t i l l contaminating the B enriched population may be sufficient and also necessary for the stimulation of B 90 TABLE VIII Lack of Inhibition of EBV Cell Line Growth WALK (DR4) Cells Antibody 3 (pg/ml) Mean CPM + SEM X Inhibition None 1339 + 289 NB-65 494 + 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) Cells None 7492 + 270 NB-65 671 + 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 NB-107 (20) 5829 + 384 22 NB-107 (5) 6341 + 1520 15 NB-107 (1.25) 5858 + 1734 22 NB-107 (0.3) 6702 + 109 11 aSee Table V for description of antibodies. TABLE IX Inhibition of Anti-p Stimulation: Purified B Cells Antibody (10 pg/ml) Mean CPM + SEM X Inhibition 969 + 85 13,225 + 156 11,379 + 724 15 12,661 + 550 5 None None0 NHL 30.5 NB-107 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 Cells Colonies* Progenitor NB-107 Exp 1 Exp 2 CFU-E 409 107 (late erythroid) + 399 110 BFU-E 204 169 (primitive erythroid) + 149 156 CFU-GM 286 128 (granulopoietic) + 254 109 CFU-G/E 1.5 1 (pluripotent) + 2 0 aNumber of colonies per 2 x 10^ normal human marrow buffy coat ce l l s . Values are means of counts from 2 replicate 1.1 ml methylcellulose cultures 5 each i n i t i a l l y containing 2 x 10 ce l l s . 93 c e l l s . If anti-LFA-1 inhibits B c e l l activation by affecting T cells and/or monocytes, and i f the contaminating T cells and monocytes are involved in the stimulation of B cells , the antibody should also inhibit the stimulation of purified B ce l l s . However, the present study clearly showed that anti-LFA-1 does not inhibit the stimulation of purified B cells by anti-p. This apparent paradox may be explained as follows: At low c e l l densities (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 latter cells are inhibited by anti-LFA-1 which results in inhibition of PBMC stimulation by anti-p. At high c e l l densities (purified B cells) anti-p stimulates B cells in the absence of accessory cells and/or monocytes perhaps due to a c r i t i c a l c e l l density which is achieved or required for endogenous release of BCGF and autostimulation (see below). The mechanisms involved in B c e l l activation are currently being actively researched. LPS stimulation of B cells appears to be macrophage but not T c e l l dependent (13). Anti-p antibody is thought to exert i t s effects on B ce 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. Proliferation of B cells in this case then requires a second signal, mediated by BCGF secreted by activated T helper c e l l s . At high concentrations (such as used in this study), anti-p has been thought to ini t i a t e a direct proliferative effect on B cells independent of T ce l l s , monocytes or their products (7,8,14). Recent studies have shown that EBV transformed B cells require either a c r i t i c a l c e l l density or supplementation with exogenous factors for proliferation. Furthermore these ce l l s appear to secrete their own autostimulatory B c e l l growth factor (17,18). Whether normal stimulated B cells at high c e l l densities are capable of autostimulation is not yet known. 94 Previous studies have not shown an inhibitory effect of anti-LFA-1 monoclonal antibodies on LPS stimulation of mouse lymphocytes (12). The differences between these and our findings may be related to species differences between mouse and human or relative concentrations of B c e l l s . Human LFA-1 expresses multiple unique antigenic epitopes that reside with varying degrees of spatial proximity to the functional region(s) of the LFA-1 molecule (19). NB-107 clearly reacts with a different epitope on the LFA-1 molecule than does TS1/18 and TS1/22 as shown by competitive inhibition studies (Table III). Monoclonal antibodies to different LFA-1 epitopes appear to vary in their a b i l i t y to inhibit T lymphocyte function (20). The region defined by NB-107 may be uniquely involved in the cooperative interaction of B cells with monocytes and/or T cells and thus susceptible to inhibition by NB-107 but not other anti-LFA-1 monoclonal antibodies. Inhibition of the MLR by NB-107 (Table VII) is similar to that previously described for other anti-LFA-1 monoclonal antibodies. In contrast, PHA stimulation of T lymphocytes was 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 is consistent with previous reports (Fig. 7,8) (6,21). LFA-1 was present on a l l EBV c e l l lines examined (9/9), but absent on 4 of 7 neoplastic B lymphoma lines. The latter finding implies the loss of LFA-1 may be somehow associated with the development of B c e l l malignancy. Loss of normal B c e l l growth regulatory c e l l surface molecules may be a step in the development of autonomous (neoplastic) c e l l growth. Alternatively, the surface expression of LFA-1 on B lymphoma cells may simply be lost by altered gene expression or product modification. The functional implications of the loss of LFA-1 on B cells are not known. 95 Although approximately half of bone marrow c e l l s are positive for LFA-1 (2) the i d e n t i t y of these c e l l s has not been established. In order to test whether LFA-1 i s important i n hematopoietic 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 c e l l s . No i n h i b i t o r y effect 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 of colonies of diverse myeloid c e l l lineages. Recently, the family of glycoproteins LFA-1, 0KM1, pl50,95 have been found to be deficient on the c e l l s of patients with chronic b a c t e r i a l infections and multiple immune abnormalities (22-24). I t has been postulated that these abnormalities result from the abnormal function of granulocytes, monocytes and/or T c e l l s and that these defective functions may be att r i b u t a b l e to the limited expression of LFA-1, 0KM1 and pl50,95 on these c e l l s . We report the i n h i b i t i o n of B lymphocyte ac t i v a t i o n by a monoclonal antibody to LFA-1, and provide evidence that this i s due to effects on monocytes and/or T c e l l s . These findings suggest that this class of c e l l surface molecules has a more diverse functional role i n the regulation of the immune response than previously realized. We are currently investigating B c e l l function and LFA-1 expression i n patients with hereditary immune deficiency and chronic b a c t e r i a l infections. 96 REFERENCES 1. Sanchez-Madrid F, Nagy J, Robbins E, Simon P, Springer TA: A human leukocyte differentiation antigen family with distinct 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 in 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: Definition of a common leukocyte cell-surface 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 cellular cytotoxicity ( 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 in 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 significance, distribution, and structure of LFA-1, LFA-2 and LFA-3: Cell surface antigens associated with CTL-target interactions. J Immunol 131: 611-616, 1983. 7. Kehrl JH, Muraguchi A, Butler JL, Falkoff RJM, Fauci AS: Human B c e l l activation proliferation and differentiation. Immunol Rev 78: 75-96, 1984. 8. Howard M, Nakanishi K, Paul WE: B c e l l growth and differentiation factors. 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 transferrin receptor blocks transferrin binding and inhibits human tumor c e l l growth in vitro. 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): Cellular distribution, 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 effects on other T and B lymphocyte functions. J Immunol 127: 590-595, 1981. 97 13. Melchers F, Corbel C: Studies on B-cell activation in vitro. Ann Immunol 134: 63-73, 1983. 14. Melchers F, Andersson J: B c e l l activation: Three steps and their variations. Cell 37: 715-720, 1984. 15. Forni L, Coutinho A: Receptor interactions on the membrane of resting and activated B cel 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 activation. In Leukocyte Typing II, 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 B lymphocytes produce B-cell 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 lines derived from Burkitt's lymphomas and other lines transformed in vitro by Epstein-Barr virus. 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): Identification of multiple antigenic epitopes and their relationship to CTL-mediated cytotoxicity. J Immunol 131: 1182-1188, 1983. 20. Springer TA: Analysis of macrophage differentiation 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 distinct antigens associated with human T-lymphocyte mediated cytolysis: LFA-1, LFA-2 and LFA-3. J Immunol 79: 7489-7493, 1982. 22. Dana N, Todd RF, Pitt J, Springer TA, Arnaout MA: Deficiency of a surface membrane glycoprotein (Mol) in man. J Clin 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 Clin 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 in boy with abnormal leukocyte function. The Lancet March 10: 535-537, 1984. v 98 C H A P T E R IV MONOCLONAL ANTIBODIES TO HLA-CLASS II DETERMINANTS: FUNCTIONAL EFFECTS ON THE ACTIVATION AND PROLIFERATION OF NORMAL AND EBV TRANSFORMED B CELLS "Probable i m p o s s i b i l i t i e s are to be preferred 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 II molecules are heterodimeric transmembrane proteins c o n s i s t i n g of a heavy (a) chain of 33-35,000 MW and a l i g h t (3) chain of 25-28,000 MW (1). Genetic and biochemical analyses of the genes and gene products encoded i n the HLA-class II (D) region have revealed the existence of three groups of molecules with s i m i l a r structures (1-3). DR i s s t r u c t u r a l l y homologous to murine I-E. DQ (DS, MB, DC) shows homology to murine I-A, and DP (SB) has been defined by primed lymphocyte typing (4). There i s evidence for at l e a s t s i x alpha and seven beta chain genes within the D region (4). At the protein l e v e l , HLA-DR homozygous c e l l l i n e s appear to express at l e a s t f i v e c l a s s II molecules: two DR molecules, two DS molecules, and one SB molecule (5). Although i t i s well established that c l a s s II genes and th e i r products are involved i n a v a r i e t y of immune responses, very l i t t l e i s known about how these molecules modulate b i o l o g i c behavior (2,3). 99 Evidence in both mouse and man indicates that class II genes determine several immunologic phenomena. These include: control of the level of immune responses, delayed type hypersensitivity, susceptibility to certain diseases, and primary (mixed lymphocyte culture) and secondary (primed lymphocyte typing) allogeneic T c e l l proliferation. In addition, HLA class II molecules are involved in T c e l l activation, apparently by playing an associative role in the recognition of antigen (3). The relative contribution of DQ vs DR molecules to these processes is unknovn. A role for HLA class II molecules in B c e l l activation has not been documented. However, accessory cells (which express class II antigens) and T ce l l s are thought to have a regulatory role in the activation of B cells (6). B cells are induced to proliferate in response to a variety of stimuli including specific 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 activation by these mitogens is not understood, although both cross-link c e l l surface molecules (6). In order to investigate the functional role of HLA class II antigens in B c e l l activation we have generated a panel of anti-class II monoclonal antibodies. In this chapter are described the characteristics of three of these, that differ in their s p e c i f i c i t i e s for DQ and DR determinants as well as in their effects on the proliferation of normal and EBV transformed B ce l l s . 2) RESULTS (A) Antibody Specificity Monoclonal antibodies to HLA class II molecules were produced using the B lymphoma c e l l line DHL-10 (DH-84, DH-224) or LPS stimulated splenic B lymphoma cells (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, respectively). In addition, these antibodies reacted with a panel of DR homozygous cells encompassing DR1 through DRw9 spec i f i c i t i e s (Table XI). Both immunoprecipitated heterodimers of approximately 35,000 and 28,000 m.w. (Fig.9). These values are similar to the molecular weights of the heterodimers immunoprecipitated by OKIa and BD-DR. NB-29 monoclonal antibody reacted with homozygous c e l l lines (HCL) DR l,2,4,6,8,w9. This pattern of reactivity is the same as that obtained with BT 3.4, a known and well characterized anti-DQ monoclonal antibody (8). NB-29 also immunoprecipitated a c e l l surface heterodimer similar in molecular weight to that precipitated by BT 3.4 and Leu-10, which is sl i g h t l y less for both chains than that precipitated by the known anti-DR monoclonals (Figs.9,10). NB-29 reacted with the same class II epitope as did BT 3.4. This was demonstrated by the ab i l i t y of BT 3.4 to completely inhibit the binding of 125 I labeled NB-29 (Table XII). NB-29 binding was also significantly inhibited 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 reactivity with DR homozygous c e l l lines and by cross-blocking studies, NB-29 appears to be a monoclonal antibody to a polymorphic determinant present on DQ molecules, that is similar or identical to that defined by BT 3.4. As shown in Table XII, DH-224 binding was blocked markedly by BD-DR but only weakly by OKIa. On the other hand DH-224 did not block either DH-84 or NB-29 binding. Removal of nearly a l l material reactive with DH-224 by "preclearing", substantially reduced the amount of DR precipitated by BD-DR. However, the amount of Leu-10 reactive material (DQ) was not affected (Fig.11). Based upon i t s reactivity with normal PBMC and DR-HCL, and from TABLE XI Reactivity of Anti-class II Monoclonal Antibodies with Homozygous DR Cell Lines: FACS Analysis 3 Specificity Antibody Cell Line D/DR HLA-ABC NB-29 BT3.4 Leu-10 1 DH-84 DH-224 BD-DR JREE DR1, DW1 A2, BW44 (W4) + + + + + + CMG DR2, DW2 A3, B7 + + + + + + RMG DR3, DW3 Al, B8 - - - + + + WALK DR4, DW4 A2, BW44, CW5 + + + + + + SWEI DRS, DW5 A29, B40 - - + + + + ELD DR6, (DW DW6 "6.1") A2, B40 + + + + + + BD DR7, DW7 A2, B13 - - - + + + MAD DR8, (LD DV8 "8.1") A2, B40, CW3 + + + + + + KOZ DRV9, , LD "4x7" AW24, 26 BVS4, 40 + + + + + + Cell lines were considered positive i f greater than 10% of ce l l s were reactive with the test antibody compared to negative control antibody of irrelevant specificity. 1 0 2 — 90 — 67 FIGURE 9 Immunoprecipitation using 1 2 5 I - l a b e l e d WALK (DR4) c e l l s . 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 p r e c i p i t a t e d by DH-84 and OKIa i s considerably less than that of the other antibodies. This probably relates to diff e r e n c e s i n antibody a f f i n i t y . The molecular weight of each chain 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). 103 FIGURE 10 Immunoprecipitation using 1 2 5 I - l a b e l e d WALK (DR4) c e l l s . 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 i d e n t i c a l bands, each of which has mobility s l i g h t l y greater than those p r e c i p i t a t e d by BD-DR. 1 0 4 TABLE XII 125 Cross Blocking of I-labeled Anti-class II Antibodies: DHL-4 Cells Labeled Antibody Blocking Antibody NB-29 DH-84 X Inhibition 0 DH-224 NHL-30.5 0 0 0 NB-65C 0 0 0 Thyl.2 c 4 3 0 NB-29 99 0 0 DH-84 78 91 0 DH-224 7 0 94 BT3.4 99 0 0 Leu-10 50 0 0 BD-DR NDa ND 72 OKIa ND ND 18 ND = not done °Calculated as described in materials and methods. °Hybridoma supernate 105 FIGURE 11 Sequential immunoprecipitation " p r e c l e a r i n g " of 1 2 5 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 s p e c i f i c i t y . DH-84, DH-224, Leu-10 and BD-DR each p r e c i p i t a t e bands of approximately 35,000 and 28,000 a.w. "B" i s precleared using DH-84 which s u b s t a n t i a l l y reduces the amount of BD-DR and DH-224. The marked diminution in the amount of material p r e c i p i t a t e d by the known anti-DR monoclonal antibody (BD-DR) ind i c a t e s DH-84 has s p e c i f i c i t y for DR molecules. The amount of Leu-10 (anti-DQ) p r e c i p i t a t e d material i s unaffected. "C" i s precleared using DH-224. In ad d i t i o n to removing material reactive with 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 pre c i p i t a t e d by BD-DR while leaving DQ r e a c t i v e material ( p r e c i p i t a t e d by Leu-10) unchanged. 106 the cross-blocking and sequential immunoprecipitation data, DH-224 appears to react with a monomorphic DR determinant similar, but not identical in specificity, to BD-DR. The specificity of DH-84 was more d i f f i c u l t to establish. DH-84 reacted with a l l HCL and normal control PBMC tested. Preclearing of c e l l lysates with DH-84 substantially diminished the amount of material reactive 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 did not substantially 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 I labeled NB-29 binding to DHL-4 cel l s . As described above, NB-29 is specific 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 reactivity with DP molecules has not been determined. (B) Inhibition of PBMC Stimulation Both DH-84 and DH-224 profoundly inhibited PBMC stimulation by antisera against IgM (mean inhibition of 70-90%), and LPS (mean inhibition of 80-90%) (Tables XIII and XIV). Titration of the inhibitory effect of DH-84 on anti-u induced stimulation showed that purified DH-84 was effective over a wide concentration range (from 4 jug/ml to 0.16 |ug/ml). Similar inhibition was not seen in cultures containing any of a number of monoclonal antibodies of unrelated specificity (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 significant inhibition of LPS or anti-u stimulation (Table XIII). Likewise DH-84 and DH-224 markedly inhibited the mixed lymphocyte response, 107 OL OL t CM 00 H e 00 O CQ X O DQ CJ Z Q CJ 2 Q FIGURE 12 Sequential immunoprecipitation " p r e c l e a r i n g " of 1 2 5 I-labeled WALK (DR4) c e l l l y s a t e . "A" i s precleared with co n t r o l antibody of unrelated s p e c i f i c i t y . NB-29 and DH-84 each p r e c i p i t a t e bands of approximately 35,000 and 28,000 m.w. "B" i s precleared with NB-29. NB-29 pr e c l e a r i n g removes a l l material reactive with i t s e l f , while not qu a n t i t a t i v e l y a f f e c t i n g the amount of material prec i p i t a t e d by DH-84. 108 TABLE XIII Inhibition of Stimulation of Normal PBMC' Mean cpm + SEM Antibody (5 ug/ml) Control Test 0 X Inhibition Anti-u d media 232 + 124 3,436 + 637 NB-29 445 + 159 2,421 + 198 17 + 10 DH-84 197 + 46 298 + 54 83 + 22 DH-224 247 + 75 939 + 397 70 + 29 LPS media 1,520 + 339 10,951 + 1 ,323 NB-29 1,541 + 296 7,969 + 237 18 + 13 DH-84 222 + 38 1,332 + 256 83 + 11 DH-224 455 + 109 2,207 + 249 84 + 11 NB-65e 523 + 53 2,914 + 484 64 + 17 Results are expressed as mean counts per minute (tjpm) + standard error of the mean (SEM) of triplicate wells containing 2 x 10 peripheral blood mononuclear cells (PBMC). Per cent inhibition was calculated as described in Materials and Methods and is the mean + SEM of values from four separate experiments. ^Cultures without mitogens. °Cultures with mitogens. ^Affi n i t y purified polyclonal goat anti-human IgM. eAntibody to transferrin receptor (hybridoma supernate). 109 TABLE XIV Inhibition of Anti-u Stimulation of Normal PBMC: Titration Using Purified Anti-HLA Class II Antibody Mean cpm + SEM Antibody Control Test X Inhibition (ug/ml) media 737 + 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-30.5a (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 NHL-30.5 is a monoclonal antibody directed against a myeloid differentiation antigen, one of several monoclonals used as negative controls. 110 while NB-29 did not, even when tested against reactive individuals (Table XV). None of these anti-class II monoclonal antibodies inhibited PHA stimulation of PBMC in culture (Table XVI). (C) Inhibition of Purified B Cells To test whether the inhibitory effects of these monoclonal antibodies were mediated via direct effects on B cells or through an action on accessory cells, B cells were extensively enriched by a combination of E-rosette depletion of T ce l l s , monocyte adherence to glass and c e l l sorting. These extensively purified populations of B cells showed an excellent stimulatory response to anti-u, and this was not affected by the addition of any of the monoclonal anti-class II antibodies studied here (Table XVII). (D) Inhibition of EBV Cell Lines To assess further whether the inhibitory effects of DH-84 and DH-224 on PBMC mitogenesis were direct or indirect, the a b i l i t y of these antibodies to inhibit the growth of EBV transformed B c e l l lines was tested. As shown in Table XVIII, both purified antibody and hybridoma supernate substantially inhibited the proliferation of EBV c e l l lines. NB-29 also inhibited these lines, but the effect 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 lines, did not inhibit their growth (Table XVIII). 3) DISCUSSION To determine whether HLA class II molecules are involved in B lymphocyte activation and proliferation, we tested several new anti-class II monoclonal antibodies for their a b i l i t y to inhibit various lymphocyte responses. Consistent inhibition of LPS and anti-^u stimulation of PBMC by two TABLE XV Inhibition of Mixed Lymphocyte Reaction Mean cpm + SEM Antibody (5 ug/ml) Control 3 Test % Inhibition 0 media 459 + 204 17,998 + 797 NB-29 591 + 118 16,117 + 1,175 13 + 3 DH-84 111 + 20 4,748 + 286 69 + 8 DH-224 139 + 69 6,654 + 644 61 + 3 NHL-30.5 679 + 141 19,810 + 1,941 0 NB-65d 343 + 93 4,028 + 502 83 + 6 Cultures without stimulator c e l l s . Cultures with stimulator c e l l s . Mean + SEM of two separate experiments. 'Hybridoma supernate. TABLE XVI Inhibition of PHA Stimulation of Normal PBMC Mean cpm + SEM Antibody Control Test X Inhibition' 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-65b 305 + 82 48,373 + 3,083 43 + 1 Mean + SEM of two individual experiments. Antibody to transferrin receptor consistently showed 40-50% inhibition of PHA stimulation. 113 TABLE XVII Inhibition of Anti-p Stimulation of Purified B C e l l s 3 Antibody (10 ug/ml) Mean cpm + SEM X Inhibition Media Control 969 + 85 Anti-p alone 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 Data is representative of three separate experiments a l l showing equivalent results. 114 TABLE XVIII Inhibition of EBV Cell Line Proliferation: ELD (DR6) Cells Antibody (5 yg/ml) Mean cpm + SEM X Inhibition 3 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 NB-65b 671 + 62 90 aData is representative of four separate experiments with each of two EBV transformed c e l l lines (ELD and WALK). ^Hybridoma supernate. 115 anti-class II 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 relative roles of DQ and DR in 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 inhibit mitogen stimulation of PBMC and purified normal B cell s , and the proliferation of EBV transformed B c e l l lines. Monoclonal antibodies DH-84 and DH-224 profoundly inhibited the anti-u and LPS stimulation of PBMC; NB-29 did not. This observation suggests that DR and DQ molecules differ in terms of their functional involvement in B c e l l activation. However, these studies could not distinguish which effects result from direct binding of the antibodies to B cells and which might result from antibody binding to accessory c e l l s . To answer this question, B cells were extensively enriched from PBMC using a combination of E-rosette separation of T cells , glass adherence removal of monocytes, and "negative" c e l l sorting. The resultant highly purified population of B c e l l s , while showing an excellent response to anti-u, was not inhibited by any of the anti-class II monoclonal antibodies studied here. These observations suggest that the anti - j j and LPS activation of PBMC depends on a DR sensitive step in which accessory cells are involved. Since DQ molecules are expressed only on the subpopulation of monocytes capable of presenting antigen (9,10), lack of inhibition by NB-29 might be due to the inabi l i t y of this monoclonal to inhibit the monocyte subpopulation which is necessary for PBMC responses to anti-u and LPS. Anti-HLA class II polyclonal or monoclonal antisera have been shown to inhibit a variety of cellular responses in human and mouse systems including: proliferative and plaque forming responses to Con A and pokeveed mitogen 116 (11-13), primary and secondary MLR (14-16), and antigen specific lymphoproliferative responses (17-19). Anti-la has also been shown to inhibit LPS stimulation of mouse lymphocytes (20). An inhibitory effect of anti-HLA class II monoclonal antibodies on anti-^u, LPS and PHA induced proliferation of human cells has not been previously reported. Anti-p antibody appears to exert i t s effects on B cells by two concentration dependent mechanisms (6). 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 (BCGF) receptors. Proliferation of B cells in this case then requires a second signal, mediated by BCGF secreted by activated helper T c e l l s . At high concentrations (as used in this study), anti-ju is thought to i n i t i a t e a direct proliferative effect on B cells independent of T c e l l s , monocytes or their products. Surface receptors for LPS on B cells appear to be distinct from surface IgM (21). Inhibition of both LPS and anti-p induced proliferation of human B cells suggests a fundamental role for HLA class II molecules in the regulation of B c e l l growth at physiological concentrations of B cells as seen in unfractionated PBMC. As a component of PBMC, B c e l l s are present at low c e l l density and may be dependent upon interaction with monocytes or a monocyte derived factor in order to proliferate in response to anti-u. At higher c e l l densities B c e l l s may not require this factor or may produce sufficient endogenous factor to be independent of accessory c e l l s , analogous to EBV transformed B cells (see below). Inhibition of MLR by anti-HLA class II antibodies as shovn in this study confirms previous reports (13). The complete lack, of inhibition of PHA stimulation suggests that activation by this mitogen, which is primarily stimulatory to T cel l s , may not involve HLA class II antigens. 117 A l l three anti-HLA class II monoclonal antibodies exerted a direct a n t i -proliferative effect on the growth of EBV c e l l lines, although inhibition was consistently greater for DH-84 and DH-224 than NB-29. Why EBV transformed c e l l lines should behave differently from purified normal B ce l l s is of interest. At low c e l l concentrations EBV c e l l lines 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 lines by the day three harvest, they were seeded at a concentration of 5 x 10 4 cells/ml or less. At this relatively low concentration, EBV transformed cells appear to be susceptible to inhibition by anti-HLA class II monoclonal antibodies. Whether this inhibitory effect can be overcome by the addition of exogenous growth factor remains to be determined. However since EBV transformed B c e l l s are not normal, the different responses of these c e l l lines compared to normal purified B c e l l s may also be due to constitutive effects resulting from EBV transformation. In summary, these findings document a diff e r e n t i a l inhibitory effect of anti-DR and anti-DQ monoclonal antibodies on the activation of PBMC by the B c e l l mitogens anti-p and LPS. Failure to inhibit the anti-^u induced stimulation of purified B cells suggests an indirect effect on accessory cell s , that at higher B c e l l densities may be overcome. Furthermore, anti-HLA class II monoclonal antibodies (anti-DQ, anti-DR and anti-DQ + DR) were shown to exert a direct antiproliferative effect on the growth of EBV transformed c e l l lines. 118 REFERENCES 1. Shackelford DA, Kaufman JF, Korman AJ, Strominger JL: HLA-HLA Class II 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 allotypic variation of human class II histocompatibility antigen a-chain 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 histocompatibility complex. Cell 36: 1-13, 1984. 4. Hunter J, Noreen H, Mickelson E, Reinsmoen N: IX International workshop: a brief summary of the cellular and serologic findings. Am Soc Histocompatibility and Immunogenetics Quarterly. Summer: 5-8, 1984. 5. Karr RW, Alber C, Goyert S, Silver 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, Fauci AS: Human B c e l l activation proliferation and differentiation. Immunol Rev 78: 75-96, 1984. 7. Howard M, Nakanishi K, Paul WE: B c e l l growth and differentiation factors. Immunol Rev 78: 185-210, 1984. 8. Brodsky FM: A matrix approach to human class II histocompatibility 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, Silver J, Stobo J: Antigen-presenting capabilities of human monocytes correlates with their expression of HLA-DS, an Ia determinant distinct from HLA-DR. J Immunol 130: 706-711, 1983. 10. Nunez G, Giles R, Ball E, Hurley C, Capra J, Stastny P: Expression of HLA-DR, MB, MT and SB antigens on human mononuclear c e l l s : identification of two phenotypically distinct monocyte populations. J Immunol 133: 1300-1306, 1984. 11. Friedman SM, Breard JM, Humphries RE, Strominger JL, Schlossman SF, Chess L: Inhibition of proliferative and plaque-forming c e l l responses by human bone-marrow-derived lymphocytes from peripheral blood by antisera to the p 23,30 antigen. Proc Natl Acad Sci USA 74: 711-715, 1977. 12. Broder S, Mann DL, Waldmann TA: Participation of suppressor T cells in the immunosuppressive activity of a heteroantiserum to human l a - l i k e antigens. J Exp Med 151: 257-262, 1980. 119 13. Mizouchi T, Yamashita T, Hamaoka T, Morinaki K: The role of Ia antigens in the activation of T cells by Con A: an evidence for the species restriction between T cells and accessory cel l s . Cell Immunol 57: 28-41, 1981. 14. Pawelec GP, Shaw S, Ziegler A, Muller C, Wernet P: Differential inhibition 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 specific for HLA-HLA Class II inhibit primary responses to HLA-D but f a i l to inhibit secondary proliferative (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 inhibitions of T lymphocyte proliferation to soluble antigen, alloantigen and mitogen by unfragmented anti I-A or anti-I-E/C monoclonal antibodies. Immunogenetics 9: 11-24, 1982. 17. Triebel F, Missenard-Leblond V, Couty M, Charron DJ, Debre P: Differential inhibition of human antigen-specific T c e l l clone proliferative responses by distinct monoclonal anti-HLA-HLA Class II 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 specific monoclonal antibodies on HLA Class II restricted lymphoproliferative responses. J Immunol 131: 2735-2740, 1983. 19. Marrack P, Kappler JW: Anti-la inhibits the activity of B cells but not a T cell-derived helper mediator. Immunogenetics 4: 541-555, 1977. 20. Niederhuber JE, Frelinger JA, Dugan E, Coutinho A, Shreffler DC: Effects of anti-la serum on mitogen responses. J Immunol 115: 1672-1677, 1975. 21. Forni L, Coutinho A: Receptor interactions on the membrane of resting 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 cells and their use in 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-cell lines derived from Burkitt's lymphomas and other lines transformed in vitro 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 virus. J Exp Med 159: 1554-1559, 1984. 25. Gordon J, Ley S, Melamed M, English L, Hughes-Jones N: Immortalized B lymphocytes produce B-cell growth factor. Nature 310: 145-147, 1984. C H A P T E R V 120 TWO MONOCLONAL ANTIBODIES THAT DEFINE UNIQUE ANTIGENIC DETERMINANTS ON B-LYMPHOMA CELLS "The thing that hath been, i t is that which shall be; and that which is done is that which shall be done: and there is no new thing under the sun." Ecclesiastes 1:9 1) INTRODUCTION Normal B lymphocytes can be induced by specific antigens or mitogens to reenter the c e l l cycle, enlarge, divide and further differentiate into immunoglobulin producing plasma cells in 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 arise in cells of B-lymphocyte lineage. The neoplastic c e l l s present in a given lymphoma represent the clonal 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 individual c e l l basis, non-Hodgkin's lymphoma cells are indistinguishable from the various stages of activated normal B lymphocytes when examined by light and electron microscopy. This is the basis of the popular concept that most of the non-Hodgkin's lymphomas are neoplasms of B lineage cells in which the large clonal population produced consists of cells that are phenotypically "frozen" or "switched-on" at some point along the B lymphocyte transformation continuum (4-8). Nevertheless, there is 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 possibility that some lymphoma c e l l s can be induced to differentiate further, and the role of abnormal gene expression in these malignancies. Recently a number of monoclonal antibodies have been evaluated as diagnostic reagents for the classification 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 specific (react only with cells of B lymphocyte lineage), B c e l l associated (react with B c e l l s but also cells of other lineages), blast associated (define antigens present on normal B-blasts but absent from resting B ce l l s ) , and antibodies whose principal reactivity i s with Burkitt's lymphoma cells or EBV transformed c e l l lines (9-24). To date, none of these have shown specificity for B lymphoma ce l l s . In this chapter, two monoclonal antibodies are described which have thus far shown remarkable speci f i c i t y for human B lymphoma ce l l s . From these i n i t i a l findings i t appears that these antibodies may be useful in the future diagnosis, classification and treatment of the non-Hodgkin's lymphomas. 2) RESULTS (A) Reactivity with Cell Lines Two monoclonal antibodies, LM-26 and LM-155, reactive with the immunizing DHL-10 lymphoma cells but not with CLL cells, were i n i t i a l l y tested for their reactivities with various c e l l lines by FACS analysis. LM-26 reacted with 4/7 B lymphoma c e l l lines; LM-155 reacted with 7/7 (Table XIX). These lines were originally derived from patients with a diagnosis of diffuse "histiocytic" lymphoma (DHL-1, DHL-4, DHL-8, DHL-10), diffuse mixed histiocytic 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 lines were unreactive with these antibodies. TABLE XIX Cell Line Reactivity of Antilymphoma Antibodies: FACS Analysis 3 C e l l line Type LM-26 LM-155 DHL-1 B-lymphoma - + DHL-4 B-lymphoma + + DHL-6 B-lymphoma + + DHL-8 B-lymphoma - + DHL-10 B-lymphoma + + U698-M B-lymphoma - + BALM-5 B-lymphoma + + JREE EBV _ _ CMG EBV - -RMG EBV -WALK EBV - w+D StfEI EBV - -ELD EBV - -BN EBV W+ -MAD EBV - -KOZ EBV - -WAY EBV - -U937 Histiocytic _ _ 1937 Histiocytic - -Jurkat T leukemia - -HL-60 Myeloid _ — K562 Erythroid - -a C e l l lines were considered positive i f >10% of cells reacted with the test antibody compared to negative control antibody of irrelevant specificity. DW=weak (10-15% of cells showed low intensity fluorescence). 123 Neoplastic c e l l lines of monocyte/macrophage (U937, 1937), T leukemia (Jurkat), myeloid (HL-60) and erythroid (K.562) origin also showed no reactivity with either antibody. Cytogenetic analysis of the Giemsa banded metaphases from non-reactive EBV c e l l line WAY-1 revealed a normal 46 X,Y karyotype. DHL-10 ce l l s , which were positive with both antibodies, had a highly abnormal karyotype: 47, XY, +7,-8, lOq", l l q + , 14q+, +M. (B) Reactivity with Fresh Tissues Eighteen of 23 non-Hodgkin's B c e l l lymphomas were positive by FACS analysis for 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 profiles respectively of ce l l s from a small cleaved c e l l lymphoma. Noteworthy is the same broad pattern of staining (small, intermediate and large cells) with anti-lambda antibodies, which establishes the presence of the neoplastic clone, and LM-26. LM-155 reacted with only 5 of the 23 B c e l l lymphomas tested thus far. Examples of B lymphoma cells with either kappa or lambda surface immunoglobulin light chains and with differing heavy chain isotypes were found to be reactive with LM-26 and LM-155. One patient with CALLA positive 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). Cells in 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 XX Analysis of Fresh Tlsauesi B Call Malignancies - Positive Pathologic13 Diagnosis % Positive PACS Patient Age/Sex Tissue8 LM-26 LM-155 K X Leu 12 Leu 5 Leu-M3 1 19P LN PB SCCL-D LSCL 79 24 5 37 79 81 1 1 78 78 26 7 2 2 2 84P LN SCCL 39 14 74 3 66 32 9 3 60P LN SCCL-P 25 1 59 <1 60 42 2 4 40P LN SCCL-D 21 <1 54 6 51 22 <1 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 LCL/B-IBS 71 32 52° 68° 71 24 2 aLN, lymph node; PB, peripheral blood; IAT, Intra-abdominal tumor; RT, retroperitoneal tumor; BM, bone marrow; PF, pleural fluid. bSCCL, small cleaved cell lymphoma; P, follicular; D, diffuse; LSCL, lymphosarcoma cell leukemia; MCL mixed small and large cell lymphoma; SNC, small non-cleaved lymphoma; LCC, large cleaved cel l lymphoma; HL, histiocytic lymphoma; ALL acute lymphoblastic leukemia; LCL, large cell lymphoma; IBS, immunoblastic sarcoma; NCL, non-cleaved cell lymphoma. °Clonal i ty 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 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 cell 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 Patnologicb Diagnosis % Positive FACS Patient Age/Sex TiBsue8 LM-26 LM-155 K X Leu 12 Leu 5 Leu-M3 1 6 OH LM SCC-F 5 <1 41 3 45 52 4 2 44H LN SCC-F <1 1 3 52 79 28 <1 3 76M LN SCC-D 4 4 4 2 60 15 3 4 74M LN SLL 3 7 61 5 72 34 6 5 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 See Table XX; T-LL, T-lymphoblastic lymphoma; PTCL, peripheral T cel l lymphoma; TCL, T cell leukemia. TABLE XXII Analysis of Freeh Tissues: Reactive Lymphoid Proliferations - Negative % Positive FACS Pathologic Patient Age/Sex Tissue Diagnosis LM-26 LM-155 K X Leu 12 Leu 5 Leu-M3 1 12F LN Lipogranulomata 2 <1 8 4 25 66 1 2 52M LN FH with KS <1 <1 13 5 24 26 4 3 76M LN RLH 7 5 30 24 44 35 5 4 77M LN MCa <1 <1 26 27 <1 <1 <1 5 10F LN FH with SH 4 1 14 8 24 62 <1 6 67F LN, submax. BLPL 7 8 50' 34 52 47 6 7 29F LN GL <1 <1 15 8 25 39 2 8 56F LN SH 4 2 14 6 25 59 2 Spleen CC 2 1 23 11 28 54 7 9 50M Spleen MCHD <1 <1 16 12 8 16 12 10 75M Lung LP I 4 <1 37 28 41 48 <1 aSee Table XX. bFH, 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. 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 after their selective activation in vitro. For this study, normal spleen cells were stimulated in culture with lipopolysaccharide (LPS), and 4 days later the cells harvested and T cells removed by rosette sedimentation with AET treated SRBC. Remaining cells consisted of 90% surface immunoglobulin positive B c e l l blasts as determined by FACS analysis of fluorescence and light scatter and morphologic observation of stained cytospins. However no evidence of any reactivity 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 cells have in most instances resulted in antibodies which detect normal B c e l l specific 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 specificity for Burkitt's lymphoma ce 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 sp e c i f i c i t y for non-Hodgkin's B lymphoma ce l l s . To our knowledge this represents the f i r s t report of antibodies with this type of reactivity. LM-26 reacted with some but not a l l B lymphoma c e l l lines, 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 lines tested. These included various c e l l lines with features of hist i o c y t i c , T leukemia, myeloid and erythroid origin. By FACS analysis, TABLE XXIII Analysis of Fresh Tissues: Miscellaneous - Negative' , FACS analysis Diagnosis Tissue 0 # cases negative Normal PB 30 Normal BM 3 Reactive lymphocytosis PB 4 BM 1 PF 1 CLL PB 10 •prolymphocyte leukemia PB 1 ALL PB 1 BM 1 MF/Sezary's PB 2 CML PB 10 BM 6 CML-BC PB 3 AML BM 7 AMML PB BM HCL PB BM Aplastic anemia PB Myelodysplasia BM aLess than 3% of cells reacted with test antibodies (LM-26, LM-155) compared to isotype identical negative control antibody of irrelevant sp e c i f i c i t y . ^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, pleural f l u i d . 131 FIGURE 15 FACS histogram of p u r i f i e d 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 c e l l s are surface immunoglobulin p o s i t i v e B c e l l s (B), which by l i g h t scatter and morphologic examination of stained cytospins, are predominantly b l a s t s . These c e l l s do not bind LM-26 (C). There i s only f i v e per cent r e s i d u a l contamination with T c e l l s (D). 132 LM-26 reacted vith 80% of B c e l l lymphomas freshly explanted from patients. No reactivity vas observed vith T c e l l lymphomas or leukcmias, CLL, hyperplastic lymph nodes, reactive peripheral blood lymphocytes, normal peripheral blood and marrov ce l l s , or blood and marrov cells from a variety of non-lymphoid hematologic malignancies. LM-155, in contrast, reacted vith a l l B lymphoma c e l l lines tested, but only veakly vith one EBV transformed B c e l l line. A l l other c e l l lines vere negative vith this antibody. LM-155 reacted vith 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 specific or lymphoma associated antigens. In the past, monoclonal antibodies vhich vere i n i t i a l l y thought to be tumor specific have turned out after extensive examination to be normal differentiation antigens expressed at lov levels or on highly restricted subpopulations of cells in normal lymphoid or myeloid tissue samples (9-24). Why large populations of malignant c e l l s accumulate that express surface antigens found only transiently on normal cells is 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 also been reported (30). Since the non-Hodgkin's lymphomas are regarded conceptually as neoplasms of mature but activated lymphocytes, ve tested the possibility that the antigens detected by LM-26 and LM-155 might also be expressed on their normal lymphoid counterparts. Hovever, FACS analysis of highly purified populations of 4 day old LPS stimulated human 133 splenic B lymphocytes failed to reveal the presence of detectable numbers of LM-26 or LM-155 positive ce l l s . Lack, of expression of antigens reactive with LM-26 and LM-155 on normal cells was further supported by the negative results obtained with a variety of reactive lymphoid hyperplasias from lymph node, peripheral blood, spleen and lung. These reactive c e l l populations contained significant proportions of in vivo activated lymphoblasts. However, ve cannot yet rule out the possibility that the antigens detected by these antibodies may s t i l l be present at low levels on minor subpopulations of normal reactive B cel l s . Since a small proportion (up to 7-8%) of reactive lymph node cells from some patients were positive by FACS analysis, i t is conceivable that these positive cells may represent a unique B c e l l subpopulation expressing these antigens. Alternatively, the detection of small numbers of positive cells in reactive lymphoid proliferations may be a sign of incipient neoplasia. Most lymphomas are clonal neoplasms and the cells present in many tumors show considerable morphologic homogeneity. However, morphologically dissimilar c e l l types may 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 particular morphologic subpopulation of cells in a given specimen. As shown in Figure 14, c e l l s from a small cleaved c e l l lymphoma that stained in a monoclonal pattern with anti-lambda light chain antisera varied in size over a considerable range including intermediate and large cells as well as small c e l l s . Similarly LM-26 also stained cells of small, intermediate and larger size. Therefore i t seems unlikely that staining patterns with this antibody are related to parameters that control c e l l size. Variations in antigen expression in different phases of the c e l l cycle is also unlikely, since in 134 most small c e l l lymphomas fever than 10% of cells are in cycle (31). Finally i t is of interest that the antigens detected by both LM-26 and LM-155 could be found on cells from B lymphomas of different morphologic subtypes, including examples composed predominantly of small or large and cleaved or noncleaved c e l l s . At present l i t t l e information about the molecular nature or function of the antigens defined by these antibodies i s available. Attempts to immunoprecipitate the antigens detected by LM-26 and LM-155 using 125 standard I c e l l surface labeling techniques (32) have to date not been successful. Hovever, i t seems unlikely that LM-26 and LM-155 detect a determinant associated vith immunoglobulin since both of these antibodies reacted vit h neoplastic cells possessing different heavy and light chain isotypes and neither reacted vith normal resting or transformed immunoglobulin positive B c e l l s . The significance of veak reactivity of each of these antibodies vith 1 of 10 EBV transformed B c e l l lines is uncertain. These c e l l lines (except for WAY-1) have been passed in culture for many years. Given the knovn genetic i n s t a b i l i t y of EBV transformed lines and the tendency for these lines to develop karyotypic abnormalities vith time (33), i t vould not be surprising i f this reactivity proved to be associated vith a genetic change that occurred after immortalization in vitro. The EBV line WAY-1 vhich vas isolated from a patient less than six months prior to testing and shovn to be karyotypically normal vas negative for LM-26 and LM-155 binding. On the other hand, the B-lymphoma c e l l line DHL-10 vhich reacted vith both LM-26 and LM-155 shoved multiple karyotypic abnormalities as has been reported for many other B lymphoma lines (34-36). Burkitt's lymphoma cells appear to express different 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 characteristic 135 chromosomal abnormalities have been described i n lymphoma c e l l s freshly removed from patients (37). This raises 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 findings suggest that many B c e l l lymphomas display on their surface common epitopes not found on the majority of resting or activated 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 result from de novo alterations of surface molecules due to other processes associated with neoplastic 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 anticipated that antibodies such as LM-26 and LM-155 w i l l be useful i n analyzing further the biology of the non-Hodgkin's lymphomas. In addition, they are po t e n t i a l l y valuable reagents for diagnosis, detection of residual disease, and possibly treatment i n future therapeutic strategies. 136 REFERENCES 1. Geha RS, Merler E: Response of human thymus-derived (T) and non-thymus derived (B) lymphocytes to mitogenic stimulation in vitro. Eur J " Immunol 4: 193-199, 1974. 2. Kehrl JH, Muraguchi A, Butler JL, Falkoff JM, Fauci AS: Human B c e l l activation proliferation and differentiation. Immunol Rev 78: 75-96, 1984. 3. Howard M, Nakanishi K, Paul WE: B c e l l growth and differentiation factors. Immunol Rev 78: 186-210, 1984. 4. Lukes RJ, Collins RD: Immunologic characterization of human malignant lymphomas. Cancer 34: 1488-1503, 1974. 5. Lukes RJ, Parker J, Taylor C, Tindle B, Cramer A, Lincoln T: Immunologic approach to non-Hodgkin lymphomas and related leukemias. Analysis of the results of multiparameter studies of 425 cases. Seminars in Hematology 15: 322-351, 1978. 6. Horning SJ, Doggett R, Warnke R, Dorfman R, Cox R, Levy R: C l i n i c a l relevance of immunologic phenotype in diffuse large c e l l lymphoma. Blood 63: 1209-1215, 1984. 7. Bloomfield C, Tajl-Peczalska K, Frizzera G, Hersey J, Goldman A: C l i n i c a l u t i l i t y of lymphocyte surface markers combined with the Lukes-Collins histologic classification in adult lymphoma. N Engl J Med 301: 512-518, 1979. 8. The non-Hodgkin's lymphoma pathologic classification project. National Cancer Institute sponsored study of classifications of non-Hodgkin's lymphomas. Cancer 49: 2112-2135, 1982. 9. Frisman D, Slovin S, Royston I, Baird S: Characterization of a monoclonal antibody that reacts with an activation antigen on human B c e l l s : reaction on mitogen stimulated blood lymphocytes and cells of normal lymph nodes. Blood 62: 1224-1229, 1983. 10. Yokochi T, Holly R, Clark E: B lymphoblast antigen (BB-1) expressed on Epstein-Barr virus activated B c e l l blasts, B lymphoblastoid c e l l lines and Burkitt's lymphomas. J Immunol 128: 823-827, 1982. 11. Zipf T, Lauzon G, Longenecker B: A monoclonal antibody detecting a 39,000 m.w. molecule that is present on B lymphocytes and chronic lymphocytic leukemia cells but is rare on acute lymphocytic leukemia blasts. J Immunol 131: 3064-3072, 1983. 12. Sugimoto T, Tatsumi E, Kemshead J, Helson L, Green A, Minowada J: Determination of c e l l surface membrane antigens common to both human neuroblastoma and leukemia-lymphoma c e l l lines by a panel of 38 monoclonal antibodies. JNCI 73: 51-57, 1984. 137 13. Longenecker B, Rahman A, Leigh J, Purser R, Greenberg A, Willans D, Keller 0, Petrik P, Thay T, Surech M, Noujaim A: Monoclonal antibody against a cryptic carbohydrate antigen of murine and human lymphocytes. Int J Cancer 33: 123-129, 1984. 14. Klein G, Ehlin-Henriksson B: Distinction between Burkitt lymphoma subgroups by monoclonal antibodies: relationships between antigen expression and type of chromosomal translocation. Int J Cancer 33: 459-463, 1984. 15. Funderud S, Lindmo T, Rund E, Marton P, Langholm R, Elgjo R, Vaage S, Lie S, Godal T: Delineation of subsets in human B-cell lymphomas by a set of monoclonal antibodies raised against B lymphoma c e l l s . Scand J Immunol 17: 161-169, 1983. 16. Klein G, Manneborg-Sandlund A, Ehlin-Henriksson B, Godal T, Wiels J, Tursz T: Expression of the BLA antigen, defined by the monoclonal 38.13 antibody, on Burkitt lymphoma lines, lymphoblastoid c e l l lines, their hybrids and other B c e l l lymphomas and leukemias. Int J Cancer 31: 535-542, 1983. 17. Lipinski M, Nudelman E, Wiels J, Parsons M: Monoclonal antibody defining a Burkitt's lymphoma-associated antigen detects carbohydrate on neutral glycolipid. J Immunol 129: 2301-2304, 1982. 18. Nadler LM, Ritz J, Hardy R, Pesando J, Schlossman S, Stashenko P: A unique c e l l surface antigen identifying lymphoid malignancies of B c e l l origin. J Clin Invest 67: 134-140, 1981. 19. Nadler LM, Stashenko P, Hardy R, Schlossman S: A monoclonal antibody defining a lymphoma-associated antigen in man. J Immunol 125: 570-577, 1980. 20. Mittler R, Talle M, Carpenter K, Rao P, Goldstein G: Generation and characterization of monoclonal antibodies reactive with human B lymphocytes. J Immunol 131: 1754-1761, 1983. 21. Jung LK, Fu SM: Selective inhibition of growth factor dependent human B c e l l proliferation by monoclonal antibody ABI to an antigen expressed by activated B c e l l s . J Exp Med 160: 1919-1924, 1984. 22. Knowles D, Tolidjian B, Marboe C, Mittler R, Talle M, Goldstein G: Distribution of antigens defined by 0KB monoclonal antibodies on benign and malignant lymphoid cells 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 differentiation. Blood 63: 1424-1433, 1984. 24. Epstein A, Morder R, Winter J, Fox R: Two new monoclonal antibodies (LN-1, LN-2) reactive in 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 nutritional requirements for the establishment and cloning of human malignant lymphoma c e l l lines. 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 lines representing different levels of maturation. Br J Cancer 47: 771-779, 1983. 27. 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Markwell M, Fox CF: Surface specific iodination of membrane proteins of viruses and eukaryotic cells using 1,3,4,6 tetrachloro-3 , 6 diphenylglycouril. Biochemistry 17: 4807-4811, 1978. 33. Steel C, Woodward M, Davidson C, Philipson J, Arthur R: Non-random chromosome gains in human lymphoblastoid c e l l lines. 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 line from a patient with a diffuse 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 line (NH-AR) from a human nodular lymphoma and a comparison with lymphoblastoid c e l l line. Cancer 46: 2438-2445, 1980. 36. Miyoshi I, Kubonishi I, Yoshimoto S, Hikita T, Dabasaki H, Tanaka T, Kimura I, Tabuchi K, Nishimoto A: Characteristics of a brain lymphoma c e l l line derived from primary intracranial lymphoma. Cancer 49: 456-459, 1982. 37. Yunis J, Oken M, Kaplan M, Ensrud K, Howe R, Theologides A: Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymphoma. N Engl J Med 307: 1231-1235, 1982. 139 C H A P T E R VI SUMMARY AND CONCLUSIONS "The youth, attracted by nature and a r t , trusts i n h i s v i v i d desire, soon to enter into the innermost sanctuary. The man r e a l i z e s , a f t e r a long peregrination that he went no further than into the propylaea." Goethe The molecular events underlying the neoplastic transformation of normal lymphocytes are unknown. A var i e t y of mechanisms have been postulated including: genetic mutation, chromosomal rearrangement, v i r a l i n f e c t i o n and inappropriate oncogene expression. The regulation of normal B-lymphocyte growth and d i f f e r e n t i a t i o n involves the i n t e r a c t i o n of key c e l l surface components with e x t r a c e l l u l a r molecules or surface components of other c e l l s . C r o s s - l i n k i n g of surface immunoglobulin or other molecules, and the i n t e r a c t i o n of receptors with growth factors provide the stimulus for B - c e l l s to enlarge, divide and d i f f e r e n t i a t e . Perturbations i n normal growth regulatory controls may r e s u l t i n autonomous p r o l i f e r a t i o n which leads to c l o n a l s e l e c t i o n , c l o n a l dominance and malignancy. C l e a r l y , through improved understanding of normal c e l l u l a r regulatory processes we may be better able to appreciate and control those events that r e s u l t i n neoplasia. The non-Hodgkin's lymphomas are a c l i n i c a l l y , morphologically and immunologically heterogeneous group of diseases. Most of these are neoplasms of B-lymphocytes. Why some B c e l l lymphomas have a rapid c l i n i c a l course while others progress only slowly i s unknown, but may r e l a t e at l e a s t i n part to the p r o l i f e r a t i v e p o t e n t i a l , tissue tropism and c e l l u l a r morphology of the 140 dominant clone within a given tumor. Attempts to predict the c l i n i c a l behavior of the lymphomas has l a r g e l y involved subtyping based on morphologic c r i t e r i a . Very l i t t l e i s known about how one morphologic subtype d i f f e r s from another i n terms of c e l l surface antigen expression and responsiveness to growth factors and other regulatory controls. The long-term goal of t h i s research was to characterize the c e l l surface antigens of B lymphoma c e l l s and to search for correlations between t h e i r expression and other f u n c t i o n a l differences d i s t i n g u i s h i n g normal and neoplastic lymphocytes. To achieve this goal a number of technical obstacles had to be overcome. One was obtaining s u f f i c i e n t numbers of lymphoma c e l l s for immunization of mice p r i o r to the production of monoclonal antibodies. A second was the development of a screening method for monoclonal antibodies that would allow the s e l e c t i o n of antibodies reactive with malignant or transformed lymphocytes but not r e s t i n g or normal c e l l s . These problems were surmounted by the use of large c e l l lymphoma c e l l l i n e s and chronic lymphocytic leukemia c e l l s as prototypes, respectively, of transformed and r e s t i n g B-lymphocytes. A t h i r d problem was how to devise methods of testing these antibodies for t h e i r a b i l i t y to detect f u n c t i o n a l l y relevant molecules. The approach taken was to screen monoclonal antibodies, raised against c e l l surface antigens, for t h e i r a b i l i t y to i n h i b i t i n v i t r o mitogen stimulation of normal B and T lymphocytes. The expression of antigens detected by these antibodies on c e l l l i n e s and f r e s h l y explanted neoplastic and non-neoplastic tissues was then examined using the fluorescence activated c e l l sorter (FACS). Based upon these approaches, t h i s thesis focuses on three areas of normal and neoplastic B c e l l function and d i f f e r e n t i a t i o n . The f i r s t two of these describe c e l l surface determinants (LFA-1, HLA-class II) which may play an important regulatory function i n normal B c e l l a c t i v a t i o n and 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 cell s . This work, is summarized as follows: Lymphocyte Function Associated Antigen (LFA-1) is Involved in B Cell  Activation Human LFA-1 is a widely expressed leukocyte antigen present on cells of myeloid and lymphoid lineage. Monoclonal antibodies to LFA-1 have been shown to inhibit in vitro T c e l l immune functions. However, a role for LFA-1 in B c e l l activation has not been documented. To investigate this possibility, we examined the distribution of LFA-1 on normal, neoplastic and EBV transformed B cells as well as the abi l i t y of a monoclonal anti-LFA-1 antibody (NB-107) to inhibit 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 identified a distinct 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 lines (N=9), but was absent from 4 of 7 neoplastic B lymphoma lines. NB-107 was observed to profoundly inhibit the response of peripheral blood mononuclear cells (PBMC) to the B c e l l mitogens anti-IgM ( u ) (mean 71% inhibition) and lipopolysaccharide (LPS) (mean 80% inhibition). In order to investigate the mechanism of inhibition, B cells were sequentially purified from PBMC using a combination of E-rosette depletion of T cells, monocyte removal by glass adherence and f i n a l l y c e l l sorting. These extensively enriched populations of B cells, while s t i l l responding to anti-p, showed no evidence of inhibition by NB-107. Growth of EBV transformed c e l l lines, cultured in the presence of NB-107, also were not inhibited by this antibody. When tested in assays for T c e l l function, NB-107 was shown to inhibit 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 cell 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 cell 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 cell 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. None of these antibodies inhibited stimulation of PBMC by PHA. 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 cell 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. LM-155 detected a determinant present on a l l 7 of these lines. 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. LM-155 reacted with one case of B cell-ALL. Neither antibody reacted with normal B cell blasts, normal peripheral blood mononuclear or marrow cells, T cell 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 cell 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 cell 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, it demonstrates for the first time a functional role for LFA-1 and HLA-class II molecules in B cell 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 be answered by future research: 1) Although it was demonstrated that monoclonal antibodies to LFA-1 and HLA-DR determinants inhibit B cell activation by acting on non-B cells, what is the exact mechanism? Do these antibodies act primarily on monocytes or T cells? Why are accessory cells or T cells important in the anti-p activation of peripheral blood mononuclear cells, but not purified B cells? 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 difficult, 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 cell 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 difficult. 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 is the function of these antigens? Are they expressed on the putative lymphoma stem cell? Further attempts at immunoprecipitating the antigens defined by LM-26 and LM-155 will 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 cell 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 cell differentiation but are lost terminally. The absence of detectable reactivity with normal B cell 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 cell 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 it 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) it 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 will 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, it 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 initial insult or perturbation to a cell 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. There is some support for this hypothesis in animal models. 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 is 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 cell, 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 cell types. The net result is the emergence of a dominant clone of cells with features characteristic of malignancy (4). There are clinical 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. The latter are associated with a more rapidly progressive clinical course. 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 its 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 it "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 its 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 its normal cellular counterpart an early committed B lymphoid stem cell; one in which immunoglobulin gene rearrangement has occurred and which expresses B cell surface markers but lacks detectable surface or cytoplasmic immunoglobulin. Likewise, the normal cellular counterpart of pre-B ALL is the pre-B cell. This cell, in addition to expressing B cell surface antigens, contains cytoplasmic but not surface IgM. B cell 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 its 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 cell 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, it is important to understand normal B cell 1 5 2 function. What factors regulate the growth, transformation and d i f f e r e n t i a t i o n of normal B c e l l s ? How do these factors i n t e r a c t with B c e l l s ? Via surface receptors? What i s the nature of these receptors? Are they perturbed i n malignant B c e l l s ? Are lymphoma c e l l s growth factor independent? Do they r e l y for the i r growth advantage on autostimulatory factors or are 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 of these factors? With respect to T c e l l s and T c e l l neoplasms some of these questions are being answered. I t i s not unreasonable to assume that the genes coding f o r B c e l l stimulatory factors and the i r receptors w i l l i n the next few years be cloned and t h e i r products characterized. Do s p e c i f i c subtypes of lymphomas correlate with a p a r t i c u l a r chromosomal abnormality or with the a c t i v a t i o n of a p a r t i c u l a r oncogene? For example, i f Burkitt's lymphoma i s caused by the a c t i v a t i o n of the myc oncogene then perhaps other morphologic subtypes of lymphomas r e s u l t from the abnormal expression of d i f f e r e n t oncogenes or expression of the same oncogene at varying times i n the c e l l cycle. What i s the nature of 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 putative lymphoma "stem c e l l " ? Does this c e l l have unique c h a r a c t e r i s t i c s that allow i t to be distinguished from associated, perhaps more d i f f e r e n t i a t e d , progeny? Why i s this c e l l unresponsive to normal growth regulatory controls? Does this c e l l d i f f e r , depending on the morphologic subtype of lymphoma or i s i t the same i n a l l B c e l l lymphomas, with other e x t r i n s i c factors contributing to the morphology of i n d i v i d u a l tumors? Nodular lymphomas have a less aggressive c l i n i c a l course than do d i f f u s e lymphomas. What factors contribute to the maintenance of th i s pattern of growth? Why i s this pattern l o s t during the progression of 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 cell 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? Spontaneous mutation? Hereditary or environmental factors? How can these factors be eliminated or controlled? Although much has been accomplished, much remains to be done. REFERENCES 154 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. Marcel Dekker Inc., New 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. Science 222: 602-603, 1983. 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 687-692, 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-cell-derived tumors in mice and men. Cell 32: 311-315, 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 non-Hodgkin'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 storey drained The wells of fancy dry, And f a i n t l y strove that weary one To put the subject by, "The rest next t i m e — " " I t i s the next time!" Thus grew the ta l e of Wonderland Thus slowly, one by one, Its quaint events were hammered o u t — And now the tale i s done, And home we steer, 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 distinction of benign and malignant breast lesions u t i l i z i n g antibody present in 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 in 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 purified anti-T antibody. Vox Sang 37: 107-110, 1979. A. Howard DR & Taylor CR. An anti-tumor antibody in 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 significance of myoepithelial c e l l staining by a tumor associated antibody. Breast, Diseases of the Breast 6: 10-15, 1980. 6. Howard DR & Batsakis JG. Cytostructural localization 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. (Letter) 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 in adults: A surgical dilemma. Arch Surg 116: 575-580, 1981. 9. Howard DR, Ferguson P & Batsakis JG. Carcinoma associated membrane antigenic alterations: Detection by lectin binding. Cancer A7: 2872-2877, 1981. 10. Howard DR. Hodgkin's disease: Pathology and pathogenesis. Crit Rev Clin Lab Sci IA: 109-131, 1981. 11. Batsakis JG & Howard DR. Sjogren's syndrome: An immune response associated disorder. Clin Lab Ann 1: 171-188, 1982. 12. Howard DR, Rundell C & Batsakis JG. Vitamin E does not modify HDL cholesterol. Am J Clin 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 for tissue histiocytes. Am J Clin Pathol 77: A01-A08, 1982. 15. Batsakis JG & Howard DR. Sjogren's syndrome: Lymphoepithelial lesion of salivary gland. ASCP Check Sample 6(7), 1982. 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 in patients with breast cancer. Cancer 51: 2053-2056, 1983. 18. Howard DR, Wicken J & Nishiyama R. Differentiation 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 clas s i f i c a t i o n and correlates. Ann Otol Rhinol Laryngol 94: 220-221, 1985. 21. Howard DR & Batsakis JG. Non-Hodgkin's lymphomas: Contemporary classification and correlates. 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 activation. In: "Leukocyte Typing II", (eds. EL Reinherz, BF Haynes, LM Nadler & ID Bernstein), Springer-Verlag, New York (in 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 (in press) 

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