Lymphocyte isolation, rosette formation, and mitogen stimulation in rhesus monkeys

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Printed in the United States

Vol. 2, pp. 539-546, 1978 Pergamon Press, Inc.

LYMPHOCYTE ISOLATION, ROSETTE FORMATION, AND MITOGEN STI~JLATION IN RHESUS MONKEYS

Diane W. Taylor, Nyven J. Marchette and Wasim A. Siddiqui Department of Tropical Medicine & Medical Microbiology University of Hawaii 3675 Kilauea Ave., Honolulu, Hawaii, 96816

ABSTRACT

Over 70% of rhesus monkey peripheral mononuclear cells were isolated on sodium metrizoate-ficoll gradients with greater than 98% purity. Rhesus blood contained 47.8% active E, 58.2% total E, and 30.2% EAC rosette forming cells. Optimal conditions for mitogen studies were determined using phytohemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and streptolysin O.

INTRODUCTION Although rhesus monkeys are frequently used as animal models for human diseases, few basic studies evaluating their cell-mediated immune (CMI) responses have been undertaken. Techniques have been developed for monitoring CMI responses of humans, but the validity of directly applying them to rhesus monkeys has not been extensively investigated. Human T cells have receptor sites for sheep red blood cells (SRBC) and form direct E rosettes; whereas human B cells and monocytes possess complement receptors and form rosettes with antibody-complement coated erythrocytes (EAC rosettes) (i). Rhesus peripheral blood lymphocytes (PBL) are reported to form E rosettes in about the same proportion as human PBL (2,3,4). Results from several studies indicate that the technique of E and EAC rosette formation may be identifying the same lymphocyte subpopulations in humans and rhesus monkeys (4,5). Various plant lectins stimulate rhesus monkey lymphocytes to undergo blast transformation, but the optimal conditions for mitogen=induced transformation of rhesus lymphocytes have only been reported for PHA (6,7). In this paper techniques for isolation of mononuclear cells, identification" of T and B cells by rosette formation and determination of in vitro mitogen responsiveness were investigated for evaluating immune responses of rhesus monkeys.

This work was supported in part by a contract (ta-C-1227) from the Agency for International Development, U.S. State Department. 0145-305X/78/0701-0539/$02.00/0 Copyright© 1978 Pergamon Press

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MATERIALS AND METHODS Animals. The 35 adult rhesus monkeys (Macaca mulatta) studied had been maintained in the University of Hawaii Primate Facility for more than a year and were free of disease as established by clinical evaluation, hematologic and serum biochemical analysis and fecal examination. Lymphocyte isolation. Three to 5 ml of heparinized blood (25 U/ml) were collected from the femoral vein of each monkey. Following centrifugation, plasma was removed, and the remaining cells were suspended in RPMI-1640 medium and layered onto Lymphoprep (sodium metrizoate-ficoll, Nyegaad and Co., Oslo, Norway). After centrifugation for 30 minutes at 1500 RPM (350 xg) at room temperature, the upper layer containing mononuclear cells was washed and the number of viable cells was determined. Slides of the isolated mononuclear cell fraction and the RBC rich pellet were prepared, fixed with methanol, and stained with Giemsa. Rosette formation. The number of E and EAC rosette forming cells (RFC) was determined by following the procedure of Auiti et al. (i) except fresh rhesus plasma (1:25 dilution) served as the source of complement for assaying monkey EAC RFC. E active and EAC RFC were counted immediately after rosette formation, whereas total E RFC were determined following overnight refrigeration. The proportion of RFC (any lymphocyte binding 3 or more SRBC) to non-RFC was determined by counting 400 lymphocytes. Mito~ens. Unless otherwise stated, mitogens were used at the following concentrations per culture: Bacto phytohemagglutinin-P (PHA), 0.3 ul and E. coli lipopolysaccharide (LPS), 330 ug, (Difco Laboratories, Detroit, MI), concanavalin A (Con A), 0.83 ug (Calbiochem, San Diego, CA), pokeweed mitogen (PWM), 1.7 ul and Streptolysin 0 (SLO), 35 ul (Grand Island Biological Co., Grand Island, NY). Lymphocyte cultures. Lymphocytes were cultured in RPMI-1640 medium supplemented with i0 mM HEPES buffer, 10% heat inactivated fetal calf serum (FCS), 2.0 mM lglutamine, i00 units/ml penicillin and I00 ug/ml streptomycin. An additional 15% fresh autologous plasma was added to each culture unless otherwise stated. In all studies, 0.15 ml of lymphocyte suspension containing 1.7 x 105 mononuclear cells was combined with 0.05 ml of mitogen in round bottom microtiter plates (Flow Laboratories, Inglewood, CA) and incubated at 37°C in a 5% CO 2 environment. Following 48 hours of incubation, 0.05 ml containing 0.7 uCi/ml tritiated methyl-thymidine (specific activity 40-60 Ci/mM, New England Nuclear, Boston, MA) was added to each well. Since preliminary experiments showed that pulse labelling for 18-24 hours resulted in maximal 3H-thymidine (3H-tdR) incorporation of mitogen treated rhesus cells, the cultures were routinely harvested after 18 hours onto filter paper discs, washed successively with 5% trichloroacetic acid, followed by methanol and dehydrated with acetone. Radioactivity was determined using a Packard tricarb scintillation counter and the counts/minute converted to disintegrations per minute (DPM) using the internal standard method with non-quenching 3H-toluene as a reference standard.

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Statistical evaluation. Values are expressed as either mean ~ standard deviation or mean standard error of the mean. For statistical analysis the data were normalized by logarithmic transformation and p values determined by student' group comparison t test. RESULTS Isolation of lymphocytes. Centrifugation of rhesus monkey cells on a Lymphoprep gradient regularly produced cell preparations containing at least 98% mononuclear cells (Table i). An average of 73.5% (range 62-90) of the mononuclear cells present in the initial blood sample was recovered. Fewer than 2% of blood mononuclear cells were sedimented with erythrocytes and neutrophils. Viability of isolated mononuclear cells always exceeded 95%.

TABLE i The Distribution of Rhesus Monkey Leukocytes Following Lymphoprep Gradient Centrifugation Percent a Mononuclear Cells Neutrophi!s

Number of Donors Upper Mononuclear layer

6

98.1 ~ 0.5

1.5 ~ 0.6

Lower Pellet b

6

1.3 + 1.0

98.4 + 1.4

a, Mean ~ standard deviation, b. Leukocytes normally discarded along with the erythrocytes.

E and EAC rosette forming cells (RFC). The proportion of E (active and total) and EAC RFC in the peripheral blood of monkeys is shown below (Table 2).

TABLE 2 Total WBC, Percent Mononuclear Cells, and Distribution of E and EAC RFC in the Peripheral Blood of Rhesus Monkeys

No. of Donors 35

a.

WBC/mm 3

% Mononuclear Cells

Active

10,680 + 4,180 a --

55.5 + Ii.0 --

47.8 + 8.6 (2,8~0) b

Mean + standard deviation,

b.

% E RFC . . Total 58.2 + 8.3 (3,4~0)

% EAC RFC 30.2 + 4.7 (I,0~0)

Average number of cells/mm 3,

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Response of rhesus iymphocytes to mito$en stimulation. The results of preliminary experiments to determine conditions for optimal lymphoblast transformation are summarized in Table 3 and Figures 1-2. The additional supplementation of the medium with homologous or heterologous serum did not significantly effect 3H-tdR uptake by unstimulated rhesus PBL (Table 3). However, homologous serum significantly enhanced the response of rhesus lymphocytes to PHA, Con A and PWM.

TABLE 3 The Effect of Plasma on Lymphocyte Responses to Mitogen Stimulation Unstimulated

PHA

ConA

PWM

Regular medium a

(i)

200+30 b

1250+_160

3260+--610

710+_90

Regular medium + 15% FCS

(2)

210~I0

1330~170

3290~360

740~360

(3)

240+_20

2250+_220

5550+~i0

1470+_180

(i) vs. (2)

NS c

NS

NS

NS

(1) vs. (3)

NS

0.01

0.01

0.01

(2) vs. (3)

NS

O.01

0.01

0.01

Regular medium + 15% autologous plasma

1.7 x 105 lymphocytes were cultured for 3 days in a. regular medium RPMI-1640 complete medium containing 10% FCS. b. Mean SEM, DPM/I06 mononuclear cells x 103. c. p values determined by group comparison t test on lOgl0 transformed data. NS = not significant at the 0.i level.

Cell concentrations between 5.0 x 104/culture to 2.7 x 105/culture had little effect on 3H-tdR incorporation (Fig. i). Although cultures containing larger numbers of cells appeared to be more responsive to Con A stimulation, the difference was not statistically significant. Maximal uptake of 3H-tdR by rhesus PBL was obtained in 3 to 5 days of incubation with PHA and Con A, and 3 to 4 days with PWM (Fig. 2). The amount of stimulation during these periods was significantly greater than that recorded for other days.

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FIG. i The effect of the number of rhesus monkey mononuclear cells/culture on lymphocyte transformation.

DAYS OF CULTURE

FIG. 2 The effect of the duration of incubation on lymphocyte responsiveness in mitogen treated cultures.

Rhesus lymphocytes were stimulated by all mitogens tested except LPS (Fig. 3). The PHA dose response curve showed that the optimal concentration of PHA for rhesus PBL was 0.30 ul/culture. Higher concentrations suppressed 3H-tdR uptake. The optimal Con A concentrations for rhesus PBL was 0.8 ug/culture. A wide range of PWM concentrations produced equivalent activation of rhesus lymphocytes. Rhesus PBL responded to high concentrations of SLO, but were unresponsive to LPS. Although considerable variation was observed between animals, all rhesus monkeys showed maximal responsiveness at the concentrations stated above.

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2,

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RHESUS MONKEYS

30

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20 onA

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PHA

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008

08

14

33

167

ug/culture

ul/culture

x 10 - 2

FIG. 3 The response of rhesus monkey lymphocytes to various concentrations of mitogens.

Mononuclear cells from 35 Macaca mulatta were tested for mitogen responsiveness under optimal cell culture conditions as described above (Table 4).

TABLE 4 The Response of Normal Rhesus Monkey Lymphocytes to Mitogen Stimulation Under Optimal Conditions

Rhesus monkeys

Unstimulated

PHA

190~20 a

3270+_220 (16.9) b

ConA

PWM

6040+--400 2690+_220 (31.1) (13.9)

a. DPM ~ SEM/106 lymphocytes x 103. b. Mean stimulation ratio (DPM in mitogen treated cultures - DPM in unstimulated cultures/DPM in unstimulated cultures).

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DISCUSSION Separation of blood cells on a Lymphoprep gradient is a suitable method for isolating rhesus mononuclear cells. This method routinely provided a cell fraction containing 98-99% mononuclear cells with high viability possessing the ability for rosette formation and blastogenesis in vitro. Routinely over 70% of all peripheral blood mononuclear cells were recovered. Isolated rhesus PBL form direct E rosettes with SRBC. Since the sum of E and EAC RFC when measured independently is approximately equal to the value obtained when EAC and E are measured simultaneously (5), it appears EAC and E RFC are monitoring 2 different lymphocyte subpopulations. Previously Malaria __et__al. (1974) and Terrell et al. (1977) respectively reported that 93.7% and 89.2% of rhesus thymocytes formed rosettes with SRBC (2,5). We have observed that E, but not EAC, rosette formation is blocked by antirhesus thymus serum, and that E RFC are not labelled by fluorescein-conjugated antirhesus F(ab)~. Thus it appears SRBC selectively bind to a subpopulation of rhesus PBL which lack easily detectable immunoglobulins and C 3 receptor sites but have thymic determinants, i.e. T cells. Ganguly et al. (1977) reported that the percentage of rhesus cells bearing immunoglobulin (24%) was the same as that forming EAC rosettes (23%) (4). Our value of 30% EAC RFC is considerably higher than those reported by Kazmieriwski et al. (3) of 8.2%, Terrell et al. (5) of 14%, and Ganguly e__!tall. (4) of 23%. In the present study, normal rhesus plasma served as a source of complement for EAC rosettes; in the other studies, mouse complement was used. Perhaps different results are obtained by using homologous and heterologous complement. In humans, active or early E rosettes are thought to represent a subpopulation of T cells that are responsible for in~nunologic surveillance and better reflect T cell competency than the total number of T cells (8). In our study rhesus monkeys averaged 47.8 ql 8.6% active E rosettes which is approximately twice the value reported for humans (28.4% (8) and 25.7%, (8). The value of monitoring active E rosettes in rhesus monkeys as an index of immunocompetency remains to be ascertained. Preliminary in vitro studies of rhesus mononuclear cells showed the importance of supplementing the media with homologous plasma. Maximal rhesus mitogenic responsiveness was only achieved in the presence of rhesus plasma; fetal calf serum could not be substituted. Mitogen dose response curves demonstrated that Con A was the most efficient stimulant for PBL of rhesus monkeys. Reed (1976) also noted that Con A was more effective than PHA and PWM for stimulating PBL of the stumptail macaque, Macaca speciosa (9). PHA and P W M w e r e also highly stimulatory for rhesus mononuclear cells. Rhesus lymphocytes responded only to high concentrations of SL0 but were not stimulated by LPS which produced transformation of mouse lymphocytes. Similar results for LPS have been reported for humans (10). Harvey et al. (1974) tested mitogen responsiveness of 4 species of nonhuman primates (baboons, Cebus, squirrel and marmosets) to PHA and PWM; however, only a single dose of each mitogen was used (ii). All 4 species were found to be less responsive to mitogen stimulation than humans. Culture conditions reported here to be optimal for rhesus monkeys differ considerably from those we have found to be optimal for human PBL° Thus investigators initiating mitogen studies using primates should be aware that use of mitogen concentrations optimal for human PBL may give erronous information about primate CMI competency. This is particularly true in studies using PHA and Con A as excessive amounts of these mitogens are suppressive for primate lymphocytes.

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ACKNOWLEDGMENTS The authors wish to thank Dr. Sidney J. Townsley for his advice in use of radioisotopes and to Ms. Jackie Green, Carolyn Willey, Nancy Nakagawa and Mr. Clem Lopez for their dedication and excellent technical assistance. REFERENCES I.

AUITI, F., CIARLA, M.V., D'AMELIO, R., and GARAFALO, J.A. Surface markers on lymphocytes of patients with infectious diseases. Infect. and Immun. 8, ii0, 1973.

2.

MALAVIYA, A.N., KUMAR, R., BHUYAN, U.N., and MOHAPATRA, L.N. Rosette forming lymphocytes: A modified technique for better stability and reproducibility; Distribution in lymphoid tissue of rhesus monkeys. Indian J. Med. Res. 62, 640, 1974.

3.

KAZMIEROWSKI, J.A., FAUCI, A.S., and REYNOLDS, HoY. Characterization of lymphocytes in bronchial lavage fluid from monkeys. J. Immunol. 116, 615, 1976.

4.

GANGULY, N.K., MOHAN, C., SAPRU, R.P., and KUMAR, M. T and B cell populations in the peripheral blood of rhesus monkeys. Int. Archs Allergy Appl. Immun. 53, 290, 1977.

5.

TERRELL, T.G., HOLMBERG, C.A., and OSBURN, B.I. Immunologic surface markers on non-human lymphocytes. Am. J. Vet. Res. 38, 503, 1977.

6.

MACKLER, B.F., AMDRAUT, A.A., WILSON, B.J., and MALLEY, A. Blastogenesis of rhesus peripheral lymphocytes with competitive summation of PHA and ALS responses. Exp. Cell Res. 71, 273, 1972.

7.

MACKLER, B.F., MALLEY, A., and ~MKRAUT, A.A. Peripheral and thoracic duct lymphocyte blastogenesis by T cell specific mitogens in the rhesus monkey. J. Med. Primat. 2, 133, 1973.

8.

KERMAN, R., SMITH, R., EZDINLI, E., and STEFANI, S. Unification and technical aspects of total T, active T and B lymphocyte. Immunol. Commun. 5, 685, 1976.

9.

REED, M.J. Studies on the peripheral blood and in vitro response of peripheral blood lymphocytes to common mitogens in Macaca speciosa. Lab. Anim. Sci. 26, 762, 1976.

I0.

CHESS, L., MACDERMOTT, R.P., and SCHOLSSMAN, S.F. Immunologic functions of isolated human lymphocyte subpopulations, i. Quantitative isolation of human T and B cells and response to mitogens0 J. Immunol. 113, 1113, 1974.

Ii.

HARVEY, J.So, JR., FELSBURG, P.J., HEBERLING, RoLo, KNIKER, W.T., and KALTER, S.S. Immunologic competence in non-human primates: Differences observed in four species. Clin. Exp. Immunol. 16, 267, 1974.