Current Topics in Microbiology and Immunology Ergebnisse der Mikrobiologie und Immunitatsforschung 66 Edited by W. Arber, Basle . R. Haas, Freiburg . W. Henle, Philadelphia· P. H. Hofschneider, J. Martinsried . H. Humphrf!Y, London· N. K. ferne, Basle . P. Koldovskj, Philadelphia H. Koprowski, Philadelphia· O. MaalfJe, Copenhagen· R. Rott, Giejfen . H. G. Schweiger, Wilhelmshaven· M. Sela, Rehovot . L. Syrucek, Prague. P. K. Vogt, Seattle E. Wecker, Wiirzburg With 24 Figures Springer-Verlag Berlin· Heidelberg. New York 1974 ISBN -13:978-3-642-65910-2 e-ISBN -1):978-3-642-65908-9 001: 10.10071978-3-642-65908-9 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. © by Springer-Verlag Berlin' Heidelberg 1974. Library of Congress Catalog Card Number 15-12910. Softcover reprint of the hardcover 1st edition 1974 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. UniversWitsdruckerei H. Stiirtz AG, Wiirzburg Table of Contents FERRONE, S., PELLEGRINO, M. A., DIERICH, M. P., and REISFELD, R. A., Expression of Histocompatibility Antigens during the Growth Cycle of Cultured Lymphoid Cells. With 10 Figures . . . . . . . . . . .. 1 BRAUN. D. G., and JATON, j.-C., Homogeneous Antibodies: Induction and Value as Probe for the Antibody Problem. With 14 Figures. 29 WINTERSBERGER, E., Nucleic Acid Synthesis in Yeast 77 Author Index 103 Subject Index 117 Expression of Histocompatibility Antigens during the Growth Cycle of Cultured Lymphoid Cells 1 S. FERRONE2, M. A. PELLEGRINO, M. P. DIERICH3 and R. A. REISFELD4 With 10 Figures Table of Contents I. Introduction. . . . . II. The Cell Life Cycle . . 2 III. Synchrony of Cell Cultures. 4 IV. Growth Cycle of Lymphoid Cells and Susceptibility to Lysis Mediated by Antisera to Histocompatibility Antigens. . . . . . . . . . . . . . . . . 5 V. Activation of the Complement System by Sensitized Cultured Lymphoid Cells during the Growth Cycle . . . . . . . . . . . . . . . . . . . . . . . 9 VI. Immunofluorescence as a Measure for the Expression of Histocompatibility Antigens on Lymphoid Cells . . . . . . . . . . . . . . . . . . 1.2 . . . VII. Growth Cycle of Lymphoid Cells and Their Absorbing Capacity for Antihisto compatibility Sera . . . . . . . . . . . . . . . . . . . . . 1. 3 . . . . VIII. Yield of Soluble Histocompatibility Antigens from Cultured Lymphoid Cells at Various Stages of Their Growth Cycle 16 IX. Discussion and Conclusions 19 References 22 I. Introduction Histocompatibility antigens are genetically determined markers which are located on plasma membranes of tissue cells of each member of a species. HL-A antigens are the gene products of the major histocompatibility locus in man and represent the human counterparts of the H-2, Ag-B, ChL-A and DL-A systems in mice, rats, chimpanzees and dogs, respectively (PALM, 1964; SNELL and STIMPFLING, 1966; RAPAPORT et al., 1970; BALNER et aI., 1971; KLEIN and SHREFFLER, 1971). The great interest in the serologic, genetic, chemical and immunological characterization of histocompatibility antigens is 1 This is publication number 793 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California. This work was supported by United States Public Health Service grants AI 10180 and AI 07007 from the National Institutes of Health and grant 70-615 from the American Heart Association, Inc., and from the California Division of the American Cancer Society Senior fellowship number D-221. 2 S.F. is a Visiting Scientist from the University of Milan, Italy. 3 M.P.D. is a Visiting Scientist supported by the Deutsche Forschungsgemeinschaft (University of Mainz), Germany. 4 Scripps Clinic and Research Foundation, Dept. of Molecular Immunology, 476 Pros pect Street, La Jolla, Cal. 92037, U.S.A. 2 s. FERRONE et al.: attributable to the fact they provide cell surface markers useful in selecting transplant donors and recipients. Although at present the role of HL-A antigens in transplantation is widely accepted, a certain degree of skepticism remains mostly among surgeons, largely because of the difficulty in predicting the fate of grafts between unrelated individuals by means of HL-A typing. The more recent interest in HL-A antigens focuses on their function in cell economy as well as on their molecular organization on cell membranes. Because of their strategic location, it appears that histocompatibility antigens may provide an excellent tool in the expanding studies directed towards the characterization of cell membranes. In this approach cultured lymphoid cells may be invaluable because they retain their histocompatibility antigen expression over long periods of time and are available in relatively large amounts (BERNOCO et aI., 1969; PAPERMASTER et aI., 1969; ROGENTINE and GERBER, 1969, 1970; KLEIN et aI., 1970; PELLEGRINO et al., 1972b). This review will discuss the data available on cell surface expression of histocompatibility antigens during the cell growth cycle and will present a critical appraisal of the techniques utilized in these studies. Studies of the expression of histocompatibility antigens during the cell growth cycle are both of theoretical and practical interest. Aside from adding to our knowledge of histocompatibility antigen metabolism, such investiga tions shed light on ordered, temporal changes in macromolecular synthesis occuring during the cell growth cycle. Changes in cell membrane structure are of particular interest since cell membranes playa major role in regulating cell proliferation and immunosurveillance alterations of the cell surface are believed to be among the major causes for the disordered proliferation of malignant cells. From the practical viewpoint, since cultured cells have become the major source of solubilized histocompatibility antigen (MANN et aI., 1968; REISFELD et al., 1970; MIYAKAWA et aI., 1971; GOTZE and REISFELD, 1974), a thorough knowledge of the expression of these antigens during the cell growth cycle aids in determining which experimental conditions achieve optimal yields. Furthermore these data will clarify whether variable results of histocompati bility antigens typing reflect the degree of reproducibility of the test system or the changing expression of these antigens during the cell growth cycle. The clinical relevance of such problems lies in the possible usefulness of cultured human lymphoid cells in detecting humoral sensitilization of prospective recipients of kidney transplant, when their sera do not react with peripheral lymphocytes in the complement dependent cytotoxic test (MORRIS et aI., 1973; FERRONE et aI., 1974). II. The Cell Life Cycle During each complete cell life cycle there is a doubling of all the structural elements and functional capacities of the nucleus and the cytoplasm. Events such as the production of ribosomes and mitochondria, chromosome reproduc tion and formation of new membranes must be coordinated by means of a Expression of Histocompatibility of Cultured Lymphoid Cells 3 variety of regulatory mechanisms and finally result in balanced growth and cell function. The pace of cell growth during the life cycle seems to be regulated mainly by events in the nucleus, i.e. chromosome replication and segregation. Thus, the analysis of these chromosomal processes is of key importance to our understanding of how the cell cycle is driven forward and how the regula tion of this process is controlled within the organism. Consequently, the sub division of the cell cycle into 4 phases reflects this approach as they are defined by what chromosomes are doing (the latter being conveniently followed by analyzing the progress of DNA synthesis). 100 M 3/4 80 E is 60 .;..c. :E ';; 40 iii! 20 o 1 2 3 4 0 4 8 12 16 20 Da,s Hours Fig. 1. The left panel illustrates the life cycle of the HeLa cells and its subdivision into four phases. The right panel depicts the relationship between the growth curve (0---0) and DNA synthesis (---) of cultured human lymphoid cells WI-L2. The data are expressed as the percentage of maximum of viable cells and of DNA synthesis The cell cycle, first recognized in bean root tips by HOWARD and PELC (1953) consists of pre- and postsynthetic gaps (G1 and G2), the DNA-synthetic period (S) and the mitotic period (M). This subdivision also forms the basis for synchrony induction techniques and provides a temporal framework upon which biochemical events can be arranged. In other words, following mitosis there is a gap in the cycle termed G during which essentially no nuclear DNA 1 synthesis takes place although near the end of this phase there is preparation for DNA synthesis (PRESCOTT, 1968) (Fig. 1). The cell life cycle can be analyzed by several techniques based on unique biochemical and physical properties of cells during specific phases of the cycle. These methods have been published in detail (STANNER and TILL, 1960; PUCK and STEFFAN, 1963; TOBEY et aI., 1966). These techniques make use either of the appearance of mitotic figures or of the occurrence of cell division. The real time or delay which is required for a perturbation to be revealed at mitosis or division is equal to the age difference between normal mitotic cells and the cells affected. For example, if a random culture is pulsed with thymidine, only S cells will be labeled. Following a time lapse equal to the duration of G 2, the first labeled cells will reach mitosis (PUCK and STEFFAN, 1963). This 4 S. FERRONE et al. : measurement can be improved by accumulating the mitotic figures with a Colcemid block. This method is tedious as scoring has to be performed visually under the microscope. An alternate method (TOBEY et aI., 1966) determines the successful com pletion of cell division by measuring total cell concentration in a Coulter counter. The logarithm of cell number in the culture proves to be a straight line when plotted against time. When at zero time an inhibitor such as excess thymidine is added which stops progress at a time in the life cycle (~) prior to m°itos is, the logarithm of cell number increases linearly for the interval T = to T = tl and then suddenly flattens out. The point of inhibition can be located exactly in the life cycle by intersection of the two above described lines. m. Synchrony of Cell Cultures In order to study the individual steps of the cycle it is necessary to syn chronize the population at some point in the cycle. This is most easily achieved when cells enter S phase. The synchrony of cell populations at this point has been accomplished by interfering with the synthesis of one or more of the deoxyriboside triphosphates which are required for synthesis of DNA, while allowing processes such as synthesis of RNA, proteins and phospholipids to proceed. Blocks are maintained for a period equivalent to G and then reversed. 1 This gathers up ,-....,70% of the cells in a logarithmically growing culture at the point of initiating DNA synthesis; the rest of the cells which were caught in the S phase remain at this point until the block is removed. In practice, synchronization can be achieved by (1) blocking the synthesis of thymidylic nucleotides with amethopterin or 5-fluorodeoxyuridine, (2) interfering with the synthesis of deoxyguanine nucleotides by adding an excess of thymidine or (3) blocking DNA synthesis with hydroxyurea (for review see MUELLER, 1969). These treatments are all maintained for a period equivalent to G and then 1 reversed. A serious limitation of these methods of synchronization is that reversible inhibitors of cell metabolism may somewhat alter the normal physio logy of the cells. To overcome these limitations another method for synchronization was used for cultured human lymphoid cells by LERNER and HODGE (1971). Using phase microscopy they found that as cultures of WI-L2 lymphocytes aged, the cells became smaller. Using DNA synthesis and viable cell counts as criteria, cell rest (stationary) phase and logarithmic growth phase could be defined in these cultures. Cultures were established at a count of 2 X 105 celis/mi. At 24 hour intervals viable cell counts were made and DNA synthesis determined by in cubating small aliquots of cells (2 ml) with 2 ,uCi of thymidine-14C. After establishment of the culture, DNA synthesis was found to be maximal at 2 days and by day 8 the rate of synthesis was approximately 2% of this maximum. Viable cell counts increased to a maximum of 6 days and then remained constant for 3 days. The 8 to 10 day old cultures containing mostly small lymphocytes not synthesizing DNA were considered to be a resting population. In order to characterize the transition from this population to active prolifera- Expression of Histocompatibility of Cultured Lymphoid Cells 5 tion in terms of the cell life cycle, resting cells were harvested, resuspended in fresh medium and monitored for DNA synthesis and mitosis. There was an 8 hour interval (G followed by DNA synthesis (S phase) and, after 18-20 1) hours, by mitosis in some cells. After 28 hours, following resuspension, 70-80% of cells treated with colchicine were arrested in metaphase. From these data it seemed apparent that resting cultured human lymphoid cells immediately entered the G phase following resuspension in fresh medium. By this approach 1 the degree of synchronization of the culture around the G phase is low. In 2 fact even a highly synchronized population of cells loses their synchrony rapidly because of individual differences in generation times and a long time is required for the cells to proceed through the G and S phases. 1 Although there are a variety of synchronization techniques available, it is worthwhile to consider certain experimental conditions to assure a favorable experimental system. Thus, reproducibility of growth rate is most important in studies which involve the timing of particular events in the cycle. Variable culture conditions, i.e. use of a variety of medium and serum supplements are a major source of non-reproducibility. Another problem stems from use of cell lines that are contaminated with mycoplasma. Since mycoplasma alters the growth rate of cells, by utilizing arginine and glutamine and cleaving deoxy nucleosides and altering patterns of nucleic acid metabolism, their presence should be determined whenever most biochemical studies are done (PETERSON et aI., 1969). However, this limitation does not really affect investigations on histocompatibility antigens since it has been shown that both short and long term infection of cultured lymphoid cells with mycoplasma do not change the quantitative and qualitative profile of HL-A antigens on cell surfaces (BRAUT BAR et aI., 1973 b). When synchronization is imposed at the point of entry of cells into DNA synthesis, only those processes synchronize which depend on or are coupled to DNA synthesis; cytoplasmic activities are not synchronized. Thus, since RNA, protein and phospholipid synthesis continue during the time when the cells are triggered for nuclear replication, a state of unbalanced growth deve lops. If this state continues too long it can result in cell death. Furthermore, the fraction of cells which are caught in S phase at the start of synchrony remain trapped at this point in nuclear replication until DNA synthesis is again allowed to go on. Such cells contribute only a small amount of synchrony. Synchrony in mammalian systems is transient, i.e. there is usually a return to a completely random log-phase growth pattern within 3-5 generations (PETERSON et aI., 1969). However, despite their limitations, synchronization procedures provide mass-cultured cells which are of considerable usefulness for the study of molecular events during the cell life cycle. IV. Growth Cycle of Lymphoid Cells and Susceptibility to Lysis Mediated by Antisera to Histocompatibility Antigens BJARING et aI. (1969) were first to report that mouse lymphoma cells showed a cyclic variation in their sensitivity to cytolytic activity of com- 6 S. FERRONE et al. : plement and H-2 antisera when incubated in vitro at 37° C for periods up to 4 hours. These results were subsequently confirmed and expanded by other investigators utilizing both non synchronized and synchronized cultures of murine lymphoid cells (CIKES, 1970; CIKES and FRIBERG, 1971; PASTERNAK et al., 1971; GOTZE et al., 1972). In these studies it was found that suscepti bility of murine lymphoid cells to H-2 antibody mediated lysis is maximal 2.0 100 --0 ........ , " , 1.6 \ \ 80 \ .i..s.... \ \ \ \ ~;;; 1.2 \or u CD c:> 0.8 x 20 o 10 20 30 40 10 20 30 40 Time IHours) Time IHours) Fig. 2. Relationship between growth cycle of L1210 cells (left panel) and susceptibility to complement dependent lysis mediated by anti H-2.4 (---) , anti H-2.28 (_._) and anti H-2.31 (-) sera (right panel). Cells derived from five cultures grown for different times were harvested on the day indicated by the arrow. The alloantisera were utilized at a dilution effecting 9S % killing of the most sensitive target cells (cells L1210 in mid log phase) during the G phase of the cell cycle, decreases during the Sand G phases 1 2 and increases again when the majority of cells divide and enter the G period 1 of the next cycle (Fig. 2). It is of interest that Moloney leukemia virus deter mined antigens on murine lymphoma cells show a similar fluctuation during the cell cycle, i.e. Moloney induced murine leukemia cells were found more susceptible to the cytotoxic effects of anti-viral antibodies and complement during the stationary than during the logarithmic phase of cell growth (CIKES and FRIBERG, 1971). Investigations with cultured human lymphoid cells have given variable results which appear to depend on the cell line investigated: cultured lymphoid cells WI-L2 (FERRONE et al., 1973; PELLEGRINO et al., 1973), RPMI 1788 and RPMI 4098 (unpublished results) do not vary in their susceptibility to lysis throughout the cell cycle, as evidenced by the fact that they elicit similar titers of HL-A alloantisera directed against antigenic determinants of the first and second segregant series, when cells in either G or S phase are utilized as 1 targets. In contradistinction, human lymphoid cells RPMI 8866 (EVERSON et al., 1973; REISFELD et al., 1974) vary in their sensitivity to HL-A alloanti sera in the complement dependent cytotoxic test, as evidenced by the fact that alloantisera titers decrease during the first 12 hours after seeding, then Expression of Histocompatibility of Cultured Lymphoid Cells 7 100 [AI [81 .8 1 80 .6 RPMI·8866 .4 ... -... .2 x .E....... . 0 .!! (AI u 1.6 1.2 .8 o .4 1 2 3 4 2 4 8 16 32 64 Days Reciprocal of Alloantiserum Dilution Fig. 3. Susceptibility of cultured human lymphoid cells WI-L2 and RPMI 8866 at various stages of their growth cycle to the lytic action of HL-A allo-antisera and absorbed rabbit complement. Panel A depicts the respective growth curves of the cell lines. Panel B illustrates their respective titration curves: the growth phase of the cells is indicated by the corresponding symbol in the growth curve (Panel A) increase in mid-log phase; however, no further change is observed when the cells reach the stationary phase. Interestingly, practically identical results are obtained using rabbit (Fig. 3), human (Fig. 4) or guinea pig complement as cytolytic reagent. Since rabbit serum contributes natural antibodies directed against a polymorphic,antigenic system present on human lymphoid cells in addition to complement components (FERRONE et al., 1971; MITTAL et al., 1973 a), this finding indicates that there is throughout the growth cycle a similar behavior of HL-A antigens as well as of those antigens against which rabbit natural antibodies are directed, at least as far as their ability to combine with antibodies and to activate complement is concerned (Fig. 5). Data from the lymphocytotoxic test alone cannot be regarded as a measure of the ex pression of antigenic determinants, since lysis of target cells depends on a complex series of interactions involving antigens, antibodies, complement components and the cell membrane (FERRONE and PELLEGRINO, 1973). Thus, the contribution of antigenic determinants to the lytic process is determined by their density, distribution and availability to combine with antibodies. Should the fluid mosaic model of membrane structure recently proposed by SINGER and NICOLSON (1972) prove to be correct, then the diffusion of antigens within the cell membrane may cause additional variability in antigen expres sion. The mobility of antigenic determinants on the membrane may even