Table Of ContentCurrent Topics in Microbiology
and Immunology
Ergebnisse der Mikrobiologie und Immunitatsforschung
66
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J.
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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
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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
