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Blastogenic response of peripheral blood lymphocytes to mitogens in cynomolgus monkeys (Macaca fascicularis).

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American Journal of Primatology 3:111-119 (1982)
Blastogenic Response of Peripheral Blood Lymphocytes
to Mitogens in Cynomolgus Monkeys ( Macaca
fascicularis )
MASASHI TATSUMI', TOORU FUJIWARA', AND MASANORI HAYAMP
'Department of Veterinary Science and 'Department of Measles Virus, National Institute of
Health, Japan
Optimal conditions for mitogen-induced transformation of cynomolgus mon
key peripheral blood lymphocytes were determined. Maximal stimulation estimated by the incorporation of tritiated thymidine ('H-TdR) occurred at the
concentrations between 25 and 50 pglml with phytohemagglutinin (PHA)and
concanavalin A (ConA), and between 50 and 100 pglml with poke weed mitogen
(PWM)in a culture volume of 120 pl containing 2 x los mononuclear cells supplemented with 10% fresh autologous serum. Lipopolysaccharide (LPS),however, could induce no significant response. In most animals examined, a high response of blastogenesis was induced by Con A, moderate by PHA, and low by
PWM. Optimal stimulation occurred after incubation for 78 hours followed by
labeling with 1 pCi of "-TdR per culture for 18 hours. Although the extent of
blastogenic response was variable in individual animals, the general pattern of
time-course and dose-response to mitogens was similar in all monkeys. With 40
monkeys tested, age nor sex did not markedly influence the extent of blastogenic response.
Key words: cynomolgus monkeys, macaca fascicularis, mitogens, lymphocyte blastogenesis, optimal conditions
INTRODUCTION
Lymphocyte blastogenesis, induced by a variety of mitogens derived from plants and
bacterias, has become an important and widely used procedure for assessing lymphocyte function both in clinical and in experimental immunology. In general, the extent of
in vitro lymphocyte blastogenesis is considered to correlate with in vivo cell-mediated
immune response, such as delayed hypersensitivity in human and guinea pig systems
[Oppenheim, 19681. As to nonhuman primates, the investigations on mitogen-induced
blastogenesis have been done with rhesus monkeys [Mackler et al., 1973; Taylor et al.,
19781, baboons [Greene et al., 1977; Eichberg et al., 19811, stumptailed monkeys [Reed,
19761,marmosets [Harvey et al., 1974; Kateley et al., 19771,cynomolgus monkeys [Humber & Hetherington, 19811, and so on.
Despite the increasing use of cynomolgus monkeys as experimental models for various
human disease, few basic studies concerning their cell-mediated immune response have
Received February 2, 1982; accepted April 13, 1982.
Address reprint requests l o Masashi Tatsumi, Department of Veterinary Science, National Instilute of Health.
4-7-1,
Gakuen. Musashi-Murayama, Tokyo 190-12, Japan.
0275-2565/82/0301-04-0111$03.00 0 1982 Alan R. Liss, Inc.
112
Tatsumi, Fujiwara, and Hayami
been undertaken. While the in vitro techniques of blastogenesis have been developed for
monitoring cellular immunity mainly in humans, the validity of directly applying them
to cynomolgus monkeys has yet to be assessed. Therefore, it seems important to establish the optimal conditions to study the function of cynomolgus monkey lymphocytes
with regard to such parameters as cell concentration, mitogen concentration, time of incubation, time of labeling with radioactive DNA precursors, and kinds of sera added to
the culture.
METHODS
Animals
Fifty cynomolgus monkeys (Macaca fascicularis) were imported from Southeast Asia,
quarantined for more than a year, and were found to be apparently healthy as judged by
clinical evaluation and fecal examination. Their age was estimated by the inspection of
dentition to be from 1 to over 5 years old.
Separation of Lymphocytes
Ten ml of heparinized blood (50 Ulml) were aseptically collected from the femoral vein
of each monkey, mixed with an equal volume of 3% gelatin solution supplemented with
0.7% NaCl and 0.2% CaCl,*2H,O, and allowed to stand at room temperature for 1hour.
Then, the leukocyte-rich fraction was collected and centrifuged at 160 g for 10 minutes.
The pellet was resuspended in 0.7 ml of Dulbecco’s phosphate buffered saline and layed
onto 0.3 ml of Lymphoprep (Sodium metrizoate-Ficoll, Nyegaad, Oslo, Norway) in a
Fisher tube. After centrifugation at 2000 g for 3 minutes in a Fisher centrifuge (Model
59, Fisher Science Co., NJ), the interface layer containing mononuclear cells was collected with a Pasteur pipette and washed twice with RPMI 1640 medium (Nissui Seiyaku Go., Tokyo, Japan). The washed cells were resuspended in culture medium to give
an appropriate cell concentration. The viability of cell suspension was usually more than
95% as determined by trypan blue dye exclusion test.
The density gradient method employed for human and other species of nonhuman primates was inadequate for cynomolgus lymphocytes because of contamination of erythrocytes which may affect lymphocyte blastogenesis. The exlent of this contamination
varied in individual monkeys, usually being more than 1001 for erythrocyte: mononuclear cell ratio. Pretreatment of heparinized blood with the gelatin solution facilitated to
obtain constant yield of pure mononuclear cells from most monkey blood by enhancing
erythrocyte sedimentation.
Preparation of Mitogens
Mitogen preparations used were as follows: Phytohemagglutinin (PHA-Y;Difco Lab.,
Detroit, MI),Concanavalin A (ConA; Pharmacia Chemical, Uppsala, Sweden),Lipopolysaccharide (LPS derived from 055:B5 E. coli; Difco Lab.), and Poke weed mitogen
(PWM;GIBCO, Grand Island, NY).The mitogens were dissolved in culture medium and
stored in small aliquots at -80°C until used.
Lymphocyte Culture
Separated lymphocytes were cultured in RPMI 1640 medium supplemented with 25
mM HEPES buffer, 100 pg/ml kanamycin, and 10% serum listed below: fresh autologous serum, inactivated autologous serum, fresh allogenic serum, inactivated allogenic
serum, inactivated male monkey pooled serum, and inactivated fetal calf serum (FCS)
(Flow Lab., McLean, VA). The monkey sera used were prepared from another blood
sample collected at the same time. A 100 pl of lymphocyte suspension at the cell concentrations, indicated in Results, were dispensed into triplicate wells of 96 flat-bottomed
microculture plates (Micro-testplate I1 # 3040, Falcon Plastics, Oxnard, CA). To each
Cynomolgus Lymphocyte Rlastogenesis
113
well was then added 20 pl of mitogens at the final concentrations in the final reaction
volume of 120 p1 indicated in the text. Lymphocyte cultures were incubated at 37°C for
24 to 120 hours in the humidified atmosphere of 5% CO, and 95% air. At a selected time
between 2 and 36 hours before the termination of culture incubation, each well received 1
pCi of 3H-TdR(specific activity 20 CilmM, Amersham Co., Buckinghamshire, England)
in 10 p1 medium. At the end of the incubation period, lymphocytes were harvested onto
glass fiber filters with the aid of a semiautomatic multiple sample harvestor (Labo
Mash, Lab0 Science Co.).The filters were washed with distilled water, dried, and placed
in scintillation vials with 5 ml scintillator. The amount of radioactivity incorporated by
lymphocytes was counted in a liquid scintillation counter (Aloka Co., Tokyo, Japan).
Counted values were expressed as mean + standard deviation (cpm).Experiments were
performed with at least more than five monkeys to determine the optimal conditions for
each parameter and representative data were presented
RESULTS
Separation of Lymphocytes
By the method described, 20 to 50 million mononuclear cells were consistently obtained without detectable contamination of erythrocytes from 10 ml of initial venous
blood from each monkey. An average of 74% (range 65-85%) of the mononuclear cells
present in the initial blood sample was recovered. The isolated mononuclear cells were
morphologically or cytochemically classified into three cell types; lymphocytes (average
87%; range 79-92%), monocytes (10%; 6-14%) andpolymorphonuclear cells (2%;0-4%).
Supplemented Sera
The effect of added serum on lymphocyte blastogenesis was studied under the condition in which cultures containing 2 x los cells were incubated with 50 pgiml of each mitogen for 78 hours followed by labeling with l pCi of "-TdR for 18 hours. As shown in
Fig. 1, the highest reactivity was obtained with 10% fresh autologous serum, whereas
relatively poor reactivity was obtained with FCS. Intermediate reactivity was induced
with other kinds of sera. Therefore, subsequent experiments were performed in the presence of 10% fresh autologous serum.
Cell Concentration
As shown in Fig. 2, there was considerable effect of cell concentration on blastogenesis
within the range of 5 x lo3to 5 x lo5cells per culture, incubated with 50 pgiml of either
PHA, Con A or PWM for 96 hours. In the case of all mitogens except LPS, 3H-TdRincorporation increased with cell concentration per culture, but started to decline after getting to maximum at 2 x lo5 cells per culture. Therefore, subsequent experiment was
performed with 2 x lo5cells per culture.
Mitogen Concentration
Optimal mitogen concentrations to obtain maximal stimulation of lymphocytes were
determined under the condition in which cultures containing 2 x lo5 cells were incubated for 78 hours in the presence of 10% fresh autologous serum followed by labeling
with 1pCi of 3H-TdRfor 18 hours. Representative data on blastogenesis of lymphocytes
from two monkeys differing in the extent of response (high and low responders) are presented in Fig. 3. Although there were considerable variations in the extent of blastogenic response of lymphocytes from individual animals (Fig. 6), the patterns of dose-response curves were in general similar in high and low responder monkeys (Fig. 3). The
optimal concentration for the stimulation of lymphocytes ranged between 25 to 50 pgiml
of PHA and Con A, and between 50 to 100 pglml of PWM. On the other hand, LPS did
not induce a significant response within the range between 0.01 to 500 pg/ml. I t was also
114
Tatsumi, Fujiwara, and Hayami
ALL0
FRESH
5
0
15
10
INCORPORATION OF 3H-TdR
/
20
CULTURE
25
( cpm X
30
)
Fig. 1. Representative data concerning the effect of serum added to the cultures. Cultures conlaining 2 x lo5
cells were incubated with mitogens for 78 hours followed by labeling with 1 WCi of '€1-TdR for 18 hours. The culture medium was supplemented to 10%with serum indicated. Each column represents lhe mean counts per minutes Icum)of 'H-TdR incoruorated into triulicate cultures of 1vmDhocvtes.
The cells of unstimulated cultures in"
_
"
cor orated less than 800 cim. Auto.: Autoiogous: Allo.:Allogenic: Inact.: Inactivated a t 56°C for 30 minutes.
P P w M :
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NUMBER OF CELLS/CULTUHt
(
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Cynomolgus Lymphocyte Blastogenesis
30 1
115
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Fig. 3. The determination of optimal mitogen concentration. Cultures containing 2 x lo5cells from two monkeys ( 0 high responder; C>low responder)were incubat,ed with mitogens for 78 hours followed by laheling with 1
pCi of IH-TdR per culture for 18hours. The culture medium was Supplemented with 10% fresh autologous serum.
Each point represents the mean counls per minute (cpm)and bar standard deviation (sd)of 'H-TdR incorporated
into triplicate cultures of lymphocytes. A: Con A; B: PHA; C PW'M; L): LPS.
demonstrated that Con A induced the highest response, PWM the lowest, and PHA
intermediate.
Incubation Time
The effect of incubation time on blastogenesis was studied under the condition in
which cultures containing 2 x lo5cells were incubated with mitogens in the presence of
10% fresh autologous serum for various periods, followed by labeling for the subsequent
18 hours. Representative results are shown in Fig. 4. Maximal incorporation of 3H-TdR
occurred after incubation for 78 hours with all mitogens except LPS. In very few animals, 3H-TdRincorporation was observed to increase up to 102 hours of incubation with
PWM. In the majority of animals, the incorporation was observed to decrease after incubation for more than 102 hours.
Labeling Time
The effect of labeling time on the uptake of 3H-TdRwas studied after cultures containing 2 x lo5cells were incubated with mitogens for 78 hours. As shown in Fig. 5, maximal
incorporation occurred after a labeling time of 18 to 24 hours with all mitogens except
Fig. 2. The effect of cell concentration on mitogen-induced hlastogenesis. 1,ymphocytes of each cell concentration per culture from one cynomolgus monkey were incubated with or without (control)mitogens for 78 hours followed by labeling with 1WCi of 'H-TdR per culture for 18 hours. The culture medium was supplemented with 10%
fresh autologous serum. Each point represents lhe mean counts per minutes Icpm) and bar standard deviation
(sd) of 'H-TdR incorporated into triplicate cultures of lymphocytes. 0 Con A; C)PHA-P; PWM; 0 control.
116
Tatsumi, Fujiwara, and Hayami
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400-
600:
c
100'
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48
I
72
lNCUBATlON TIME
96
(
120
hr )
Fig. 4. The determination of optimal incubation time. Culture containing 2 x 10' cells from onc cynomolgus
monkey were incubated with or withouL (control)mitogens for indicated periods followed by labeling wiLh 1&i of
'H-TdR per culture for the last 18 hours. The culture medium was supplemented with 1070fresh autologous s t ~
rum. Each point represents the mean count per minute (cpm)and bar standard deviation (sd)of "H-TdRincorporated into triplicate cultures of lymphocytes. 0 Con A; 0 PIIA-P; PWM; Ccontrol.
10-
-
1
2 4 6 1 2
18
24
TIME AFTER 'H-TDR ADDlTlON
36
( hr
1
Cynomolgus Lymphocyte Blastogenesis
70
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Fig. 6 . The effect of age or sex on mitogen-induced hlastogenesis. Under the optimal assay conditions determined, cultilres containing 2 x 10' cells from 40 cynomolgus monkeys were incubated with mitogens for 78
hours followed by labeling with 1pCi of 3H-TdRper culture for 18 hours. The culture medium was supplemented
with 1070fresh autologous serum. Each point represents the mean counts per minute (cpm)of 'H-TdR incorporated into triplicate cultures of lymphocytes from individual monkey. F: female: M: male: 0 Con A (male); 0 Con
A (female);'2PHA (male);WPHA (female).
LPS. Longer labeling time led not only to the decrease of specific incorporation of
"H-TdR, but also to the increase of background incorporation.
Age or Sex Difference
The effect of age or sex of donor animals on blastogenesis was studied with 20 male and
20 female monkeys under the optimal assay conditions described above; i.e., cultures
containing 2 x lo5cells were incubated for 78 hours with optimal dose of each mitogen
in the presence of 10% fresh autologous serum and were labeled for 18 hours with 1pCi
of "-TdR. Although the extent of blastogenic response varied from one animal to the
other, it did not seem to be correlated significantly with age nor sex of animals.
Fig. 5 , The effect of labeling time on mitogen-induced blastogenesis. Cultures containing 2 x los cells from one
cynomolgus monkey were incubated with or without (control)rnitogens for 78 hours followed by labeling with 1
&i of 'H-'!.'dR per culture for indicated periods. The culture medium was supplemented with 10% fresh autologous serum. Each point represents the mean counts per minute (cpm)and bar standard deviation Isd)of 'H-TdR
incorporated into triplicate cultures of lymphocytes. 0 Con A; 0 PHA-P; W PWM; U control.
118
Tatsumi, Fujiwara, and Hayami
DISCUSSION
Because of the increasing interest in the use of the lymphocyte transformation test in
clinical and experimental immunology, it is necessary to establish a reliable and reproducible culture system. However, there have been only limited numbers of reports on
standardization of cynomolgus lymphocyte culture system [Humber & Hetherington,
19811. In the present study, we have established the optimal assay conditions to obtain
maximal stimulation of cynomolgus lymphocytes with mitogens using separated lymphocytes in a microculture system. A microculture system has several advantages over
macro and semimacroculture systems previously reported in the smaller amount of
blood and other reagents, in addition to the ease of setting up larger number of replicate
cultures [Kateley et al., 1977; Mackler et al., 1973; Reed, 19761. Recently, Humber and
Hetherington 119811reported a micro method of blastogenesis in cynomolgus monkey
lymphocytes using a whole blood culture system. However, for the purpose of studying
the effect of cellular interactions and serum factors on blastogenic response, the use of
lymphocytes separated from other blood elements as responding cells to mitogens may
be more suitable than that of whole blood culture. Nonspecific influence of erythrocytes
on blastogenic response should also be considered in a whole blood culture system [Luquetli & Janossy, 1976;Mackler et al., 19721. Although the widely used method of separating blood cells by density gradient as described by Boyum [1968]is suitable for separating mononuclear cells from peripheral blood of human and other species of nonhuman
primates such as rhesus monkeys [Taylor et al., 19781, baboons [Greene et al., 1977;
Eichberg et al., 19811, and marmosets [Kateley et al., 19771it is unsuitable for separating
cynomolgus lymphocytes because of considerable contamination of erythrocytes. Application of gelatin solution made it possible to avoid this contamination resulting in constant and adequate yield of mononuclear cells from cynomolgus peripheral blood.
The present study has demonstrated that cynomolgus lymphocytes were stimulated
by Con A, PHA, and PWM, but not by LPS in a similar manner to that reported in other
primates including humans. In cynomolgus, as well as rhesus monkeys [Taylor et al.,
19781,the most effective stimulant for peripheral blood lymphocytes was Con A which is
reported to affect predominantly T cells in mice [Greaves & Janossy, 19721. PHA and
PWM also stimulated cynomolgus lymphocytes but to a lesser extent than Con A. However, in the case of humans, PHA has been shown to be the most effective mitogenic
stimulant [Douglass et al., 19691. In humans, PHA and Con A are predominantly T cell
inducers and PWM may stimulate some U and some T cells. The cell populations stimulated by these mitogens has not been clearly defined in Macaca species. Lack of blastogenic response to LPS which stimulates mouse B cells to differentiate into immunoglobulin-secreting cells was reported also in humans [Chess et al., 19741 and nonhuman primates, such as rhesus monkeys [Taylor et al., 19781and marmosets [Kateley et al., 19771.
The optimal dose of PHA or Con A obtained here for blastogenic response in cynomolg u s lymphocytes was similar to that in baboons [Greene et al., 19771, but higher than
that reported for rhesus monkeys [Taylor et al., 19781 and cynomolgus monkeys [Humber & Hetherington, 19811. The optimal dose of PHA, Con A, and PWM for maximal
stimulation of cynomolgus lymphocytes was much higher than that of humans [Douglass et al., 19691. We also confirmed that the optimal dose was approximately tenfold
higher for cynomolgus lymphocytes than for humans (data not shown).
Kinetics of blastogenic response of cynomolgus lymphocytes was similar pattern in human and other species of nonhuman primates reported by other investigators. SOfar as a
limited number of animals were examined in the present study, there seemed to be no
significant relationship between the extent of blastogenic response and age or sex of animals. We are currently investigating whether or not the blastogenic response of individual monkey reflects its immunological response to particular antigens.
Cynomolgus Lymphocyte Blastogenesis
119
CONCLUSIONS
1.Optimal a s s a y conditions for mitogen-inducedblastogenesis of cynomolgus monkey
lymphocytes were determined; i.e., cultures containing 2 x lo5cells were incubated w i t h
mitogens for 7 8 hours in the presence of 10% fresh autologous serum followed b y labeli n g with 1 pCi of "-TdR for 18 hours.
2. O p t i m a l mitogen dose for cynomolgus lymphocytes ranged between 2 5 and 50 pglml
w i t h PHA and Con A, and between 50 and 100 pglml w i t h PWM. LPS did not induce a
significant response in cynomolgus lymphocytes.
3. Age or sex of donor animals did not markedly influence the e x t e n t of blastogenic
response.
ACKNOWLEDGMENTS
T h i s work was p a r t l y supported by a Grant-in-Aid for the E n c o u r a g e m e n t of Young
Scientist, no. 577971 (1980),f r o m t h e M i n i s t r y of Education, Science and Culture, Japan. The authors would like to t h a n k Drs. A. Sugiura and A. Yamada, Department of
Measles Virus, NIH J a p a n , for their valuable discussion and critical r e a d i n g i n preparation of t h i s paper.
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