A method to assess the proliferative activity of small numbers of murine peripheral blood mononuclear cells

Journal oflmmunologicalMethods, 96 (1987) 171-177

171

Elsevier JIM04203

A method to assess the proliferative activity of small numbers of murine peripheral blood mononuclear cells * W i l l i a m J. P e t e r s o n Geriatric Research, Education and Clinical Center (GRECC), West Los Angeles VA Medical Center, Los Angeles, CA 90073, and Department of Medicine, UCLA, Los Angeles, CA 90024, U.S.A.

(Received 6 June 1986, revised received 11 September 1986, accepted 29 September 1986)

An economical, reproducible method was developed to assess the mitogenic response of small numbers of murine peripheral blood mononuclear cells (PBMC, 103/ml) to phytohemagglutinin (PHA) stimulation in the presence of feeder layer cells (FLC) and interleukin-2 (IL-2). A linear cell dose-mitogenic response relationship was then demonstrated between the activity of PBMC and spleen cells of individual mice (slope = 1.054 + 0.037; r = 0.989 + 0.003). This would suggest that mice can be used for longitudinal studies because small blood volumes can be obtained frequently over a period of time from individual mice and the PBMC can be assessed for their T cell proliferative activity. Key words: Lymphocyte, mouse; T cell; Mitogen response

Introduction

Over the past 35 years, the mouse has been established as a superior animal model to study various immunological activities. However, one limitation is that the mouse has not been satisfactorily adapted for longitudinal studies. That is, there is no method that would enable one to assess small blood volume samples from individual mice over a period of time and for an immunologic index that is reflective of individual mice. Consequently, investigators performing time-dependent immunologic studies have been required to kill mice at various time intervals to assess their immune status (i.e., perform cross-sectional studies). Suffice it to say, cross-sectional studies require a relatively large sample size per time interval to minimize the variation between individuals sam-

pied at different time points. In light of the increasing interest in evaluating the delayed effects of various exogenous perturbants including diet, radiation, carcinogens, etc., and of endogenous perturbants including slow viruses, senescence, etc., and in light of the mounting mouse budget, studies were carried out to determine whether small blood volumes from mice can be processed for quantitative immunological assessment. The results presented here show that (a) the PBMC processed from small volumes of blood can be assessed in a quantitative manner for their mitogenic responsiveness to P H A stimulation by supplementing them with F L C and IL-2 and (b) the PBMC response is linearly correlated with that of the spleen in individual mice.

Materials and methods Correspondence to: W.J. Peterson, Geriatric Research, Education and Clinical Center (GRECC), West Los Angeles VA Medical Center, Los Angeles, CA 90073, U.S.A. * Supported by V.A. Medical Research Funds.

Animals

C 5 7 B L / 6 N N i a mice (3-5 months of age) were purchased from Charles River Breeding Laborato-

0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

172 ries (Wilmington, MA) through the National Institute of Aging and maintained (five or less per cage) in laminar flow racks (Lab Products, Rochell, N J) and allowed free access to food (Purina Autoclavable Rodent Chow) and acidified water (pH 2.5, 1 N HC1).

Reagents Rabbit antithymocyte serum (ATS, Microbiological Associates, Bethesda, MD) was decomplemented by heating at 56°C for 30 min, diluted 1 : 3 with phosphate-buffered saline (PBS, UCLA Media Service) absorbed once with agarose (Seaplaque, FMC Corporation, Rockland, ME), centrifuged (TJ-6R, Beckman Instruments, Palo Alto, CA) at 1520 x g for 10 rain, and the supernatant was removed and absorbed once with an equal volume of packed mouse RBC, and centrifuged again. The final supernatant was filtered through a 0.45 ~m Nalgene filter (Nalge Company, Division of Sybron Corporation, Rochester, NY) and stored ( - 70 ° C) until needed. Guinea pig complement (C', Flow Laboratories, McLean, VA, or Accurate Chemical Co., Westbury, NY) was reconstituted according to instructions provided by the vendor, diluted 1 : 3 with PBS, absorbed once with agarose, centrifuged and the supernatant was removed, sterilized by filtration, and stored frozen ( - 70 ° C) until needed. Purified IL-2 (rat origin) was purchased commercially (Collaborative Research, Lexington, MA) in lyophilized form and reconstituted with RPMI 1640 containing 25 mM Hepes, 0.1% gentamicin sulfate (50 mg, Sigma Chemical Co., St. Louis, MO) and 10% fetal bovine serum (FBS, Hyclone, Logan, UT). There were not any known factors in this preparation that would have influenced [3H]TdR incorporation.

Cell suspensions PBMC were obtained by bleeding individual mice from their retroorbital sinus with a pasteur pipette containing 0.5 ml of 0.2 M sodium citrate. The blood was dispensed into 12 × 75 mm plastic tubes (Falcon, Division of Becton, Dickinson and Co., Cockeysville, MD) containing 0.5 ml of sodium citrate, diluted 1:2 with PBS, layered on an equal volume of Lympholyte-M (Accurate Chemical Co.) and centrifuged (room tempera-

ture) at 400 × g for 30 min. The white cells at the interface were collected in a pasteur pipette, dispensed into tubes, washed three times and resuspended in medium containing RPMI 1640 supplemented with 25 mM Hepes, 10% FBS, 1% Lglutamine (200 mM, Flow Laboratories) and 5 × 10 -5 M 2-mercaptoethanol (2-ME, Sigma Chemical Co.), counted, the viability determined by trypan blue exclusion, and the concentration adjusted appropriately. For single animal assessment, 250 #1 of blood containing 1.27 × 10 6 (_+ 0.219 SEM) white cells was collected from each mouse. The yield of PBMC after separation on Lympholyte-M was 0.57N106 (_+0.10 SEM) PBMC. This represented a recovery of 45%. The loss of cells was not important because Lympholyte-M is a nonselective separation medium. To perform multiple experimental analysis, some mice were bled to death and PBMC samples were pooled to obtain enough cells. Spleens were obtained from the PBMC donors following decapitation. Cell suspensions (SC) were prepared by teasing individual spleens in medium, washing the cells once by centrifugation, resuspending in medium and filtering through sterile wire screen (200 mesh, Cambridge Wire Cloth Co., Cambridge, MD). In some experiments spleen cells were pooled to obtain enough cells for multiple experimental analysis. The cells were then counted, the viability determined, and the concentration adjusted appropriately. FLC were prepared by teasing spleen cells in medium without 2-ME and exposing them to 4000 rad of radiation to inhibit cell division (Casarett, 1968). Medium without 2-ME was used in this step because sulfhydryl compounds can act as radioprotective agents (Casarett, 1968). Contaminating T cells were then removed by mixing cells from one spleen in 3 ml of medium, with an equal volume of ATS and C' and incubating the mixture at 37°C for 1 h. The cells were then washed three times, resuspended in medium containing 2-ME, filtered and counted.

Mitogenesis Cell dose-response studies were performed by stimulating PBMC and SC from the same donor with PHA (1.25 /~g/ml, Wellcome Laboratories, Beckenham, England) in the presence or absence

173

of FLC and IL-2 in Microtest III plates (Falcon). The cultures were incubated in a CO 2 incubator (37°C, 5% CO z in humidified air) for 56 h, pulsed with [3H]thymidine ([3H]TdR, 0.5 /~Ci, specific activity 6.7 C i / m M , New England Nuclear Co., Boston, MA), incubated an additional 16 h before the cultures were harvested onto glass fiber filters with an automatic cell culture harvesting apparatus (Brandel, Rockville, MD). The sample filters were placed in shell vials (Fisher, Tustin, CA), dried and covered with 1 ml of scintillation fluid (Scintiverse, Fisher, Tustin, CA) (Peterson, 1982). The vials were capped, placed inside a standard scintillation vial and counted in a liquid scintillation counter (LS-350, Beckman, Irvine, CA).

Statistical analysis The data were processed by log transformation (Zeigler, 1974), and the results are reported as the mean + the standard error of the mean (SEM). Slope analysis was performed by fitting regression lines to the data by the least-squares method (Remington and Schork, 1970). The coefficient of correlation and the intercept were determined for all regression lines.

Results

Determination of the time-dependent pattern of PHA-induced mitogenic response of PBMC and SC The response of PBMC differed from SC in terms of the time of peak activity (Fig. 1). Maximum activity was obtained between 40 and 56 h for PBMC, and between 56 and 72 h for SC. Therefore, in the first series of experiments, the mitogenic response of small and large numbers (1.9-7.8 x 1 0 4 / m l ) of pooled PMBC and SC to PHA stimulation was determined in the presence of increasing doses of FLC (0-5 x 106/ml) and constant excess concentration of IL-2. The results (Tables I and II) showed that maximum response of both PBMC and SC occurred in the presence of 0.625-1.25 × 106 F L C / m l . This confirms our previous results with SC (Peterson, 1982) and suggests that lymphoid cells from various organs, irrespective of the inoculum size, respond vigorously when they are supplemented with an optimum dose of FLC.

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Fig. 1. Comparison of the response profile of pooled PBMC and SC. ( . . . . . . ), PBMC; ( ), SC; (©, zx), cultures were pulsed and harvested every 8 h; (e, A), cultures were pulsed every 8 h and harvested 16 h later; cell dose, 1 - 2 x 1 0 6 / m l ; vertical bars, SEM of triplicate samples. In most cases the SEM was too small to be illustrated.

In the second series of experiments, IL-2 was titrated in terms of half maximal units to reduce the slope from 2 to 1 of a log spleen cell dose-log response curve in the presence of an excess of FLC (1.25 x 1 0 6 / m l ) . Reduction of the slope by 1 U indicates saturation by IL-2. The results (Table III) showed that 3.5 half maximal units/ml is the minimum concentration of IL-2 necessary to maximally change the slope. Fig. 2 demonstrates graphically how the slope of the log spleen cell dose-log response curve can be changed from 3 to 2 by supplementing the spleen cell cultures with FLC (1.25 x 106/II11) and from 2 to 1 by supplementing the spleen cell cultures with

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Fig. 2. Effect of FLC and IL-2 on the slope of log spleen cell dose-log response curves of pooled SC. (O), SC only; (e), S C + F L C (1.25×106/ml); ( ~ ) , S C + F L C ( 1 . 2 5 x 1 0 6 / m l ) + IL-2 (13.92 half maximal units/ml).

174 TABLE I THE RESPONSE OF PBMC TO PHA IN THE PRESENCE OF EXCESS IL-2 AND INCREASING CONCENTRATION OF FLC FLC concentration ( x 10 6/ml)

PBMC concentration (× 10 4/ml) 1.9 3.9

0 0.16 0.31 0.62 1.25 2.50 5.00

1852 (2050,1673) 3237 (3449, 3038) 4076 (4265, 3895) 3650 (3697, 3604) 4004 (4354, 3682) 2787 (2880, 2697) 1 593 a (1684, 1 506)

7.8

5364 7812 7961 7296 7145 5418 3 327

(5 886, 4889) (8214, 7429) (8694, 7289) (7813, 6813) (7908, 6455) (5635, 5209) (3 509, 3 154)

10224 11 121 12910 16175 16728 12445 6 322

(10495, 9968) (11552, 10707) (14675, 11358) (17318, 15107) (18578, 15063) (12971, 11 939) ( 6 740, 5 930)

a Values are 3 standard deviations (SD) above background counts; the range of background counts is 55-103, 100-233 and 121-253 cpm for cultures containing 1.9, 3.9 and 7.8 x 104 pooled PBMC, respectively. The response of FLC in the presence of excess IL-2 ranges from 60 to 69 cpm per culture. The concentration of IL-2 used in this study was 3.48 half maximal units/ml. The figures in parentheses represent the mean plus or minus the SEM.

TABLE II THE RESPONSE OF SC TO PHA IN THE PRESENCE OF EXCESS IL-2 AND INCREASING CONCENTRATION OF FLC FLC concentration (× l0 6/ml)

SC concentration (× 10 4)

0 0.16 0.31 0.62 1.25 2.50 5.00

439 1954 2220 2611 2288 1684 954

1.9

3.9 (507, 379) (2003, 1907) (2362, 2087) (2723, 2503) (2434, 2151) (1776,1597) (978, 930)

1614 4223 4319 4764 4697 3680 1927

7.8 (1 761, 1479) (4681, 3809) (4623, 4034) (4942, 4592) (4956, 4451) (3776, 3586) (1 986, 1870)

4351 (4461, 4244) 8165 (8508, 7835) 9762 (9959, 9569) 9092 (9474, 8724) 9881 (10040, 9724) 7844 (8114, 7583) 4014 (4162, 3 872)

a Values are 3 SD above background counts; the range of background counts is 77-103, 69-176 and 70-192 cpm for cultures containing 1.9, 3.9 and 7.8 × 104 pooled SC, respectively. The response of FLC in the presence of excess IL-2 ranges from 60 to 69 cpm per culture. The concentration of IL-2 used in this study was 3.48 half maximal units/ml. The figures in parentheses represent the mean plus or minus the SEM.

TABLE III

F L C (1.25 x 1 0 6 / m l ) a n d I L - 2 (13.93 h a l f u n i t s /

EFFECT OF IL-2 CONCENTRATION ON THE SLOPE OF THE SC DOSE-RESPONSE CURVE

ml).

Number of half maximal units of IL-2/ml a

Slope

0.00 0.87 1.74 3.48 6.97 13.93

2.02 + 0.29 2.03_+0.219 1.58+0.20 1.16+_0.14 1.07+0.15 1.00_+0.13

a IL-2 was titrated in cultures containing an excess constant dose of FLC (1.25 x 106/ml) and increasing doses of pooled SC. SC ranged from 4.5x104 to 8x105/ml.

Response patterns of PBMC and SC in the presence of FLC and IL-2 The mitogenic response of varying doses of p o o l e d P B M C a n d S C ( 1 - 6 2 5 x 10 3 ) w a s d e t e r m i n e d i n t h e p r e s e n c e o f F L C (1.25 x 1 0 6 / m l ) a n d I L - 2 (3.5 h a l f m a x i m a l u n i t s / m l ) . T h e r e s u l t s , w h i c h are s u m m a r i z e d i n T a b l e s I V a n d V, y i e l d e d t h r e e i n t e r e s t i n g findings. The first a n d m o s t striki n g f i n d i n g is t h e d e m o n s t r a t i o n o f a m a r k e d enhancing effect of the two supplements on the mitogenic response of PBMC and SC over a wide

175 TABLE IV THE MITOGENIC RESPONSE OF PBMC TO PHA IN THE PRESENCE OF FLC AND IL-2 Cell dose x 1 0 3/ml

Medium

1 2 5 10 19 39 78 156 312 625

144 a (187, 111) 66 ~ (130, 33) 80 ~ (95, 67) 83 a (100, 70) 84 a (100, 71) 89 a (107, 73) 110 ~ (125, 96) 211 (242, 184) 563 (642, 495) 5344 b (6386, 4472)

Supplementation of PBMC FLC

IL-2

FLC and IL-2

84 a (91, 76) 100 ~ (111, 89) 5 (19, 1) 52 (74, 36) 128 (143,115) 275 (290, 261) 528 (581,480) 1462 (1626, 1 314) 4248 (4590, 3932) 33237 (34246, 32258)

142 (179, 113) 70 (84, 58) 260 (316, 214) 883 (1008, 773) 2065 (2142, 1 991) 4513 (4889, 4167) 10782 (11615, 10008) 20 920 (21 192, 20 653) 54190 (54927, 53463) 88556 (94883, 82651)

181 (257, 128) 397 (466, 338) 1 121 (1233, 1020) 1902 (1 959, 1847) 3 816 (3 993, 3 648) 8222 (8538, 7917) 17094 (17419, 16 774) 34 319 (36 500, 32 268) 53384 (54283, 52499) 84794 (87498, 82173)

a Uncorrected values (cpm of stimulated cultures not corrected by subtraction of background counts). b Values are 3 SD above background counts; the range of background counts is 93-183, 81-138, 85-607 and 79-470 cpm for cultures of pooled cells supplemented with medium, FLC, IL-2 and FLC and IL-2, respectively. The figures in parentheses represent the mean plus or minus the SEM.

TABLE V THE MITOGENIC RESPONSE OF SC TO PHA IN THE PRESENCE OF FLC AND IL-2 Cell dose x 10 3/ml

Medium

1 2 5 10 19 39 78 156 312 625

62 (88, 44) 67 (93, 48) 12 a (44, 3) 117 a (126, 109) 126 a (198, 81) 26 a (36, 19) 111 a (131, 95) 68 (103, 46) 355 (376, 336) 5 315 b (5 733, 4 928)

Supplementation of SC FLC

IL-2

FLC and IL-2

136 a (204, 91) 266 a (436, 163) 173 a (186, 160) 98 (140, 69) 99 (138, 70) 159 (208, 122) 358 (413,361) 1144 (1 235, 1060) 5 819 (6055, 5 592) 31 388 (32 611, 30 210)

71 (85, 60) 114 a (151, 86) 36 (75, 17) 147 (259, 84) 696 (836, 580) 1 827 (2269, 1471) 5676 (5919, 5443) 12 296 (12 597, 12 001) 28267 (29918, 26700) 53 792 (55 986, 51 686)

34 (210, 6) 226 (344, 139) 610 (751,492) 1 153 (1 217, 1092) 2328 (2399, 2260) 4710 (4897, 4530) 10060 (10583, 9564) 21 977 (23 358, 20678) 49551 (52333, 46918) 83 172 (85 087, 81 299)

a Uncorrected values (cpm of stimulated cultures not corrected by subtraction of background counts). b Values are 3 SD above background counts; the range of background counts is 12-176, 62-186, 76-1551 and 101-954 cpm for cultures of pooled cells supplemented with medium, FLC, IL-2 and FLC and IL-2, respectively. The figures in parentheses represent the mean plus or minus the SEM.

r a n g e of cell d o s e ( P B M C , 2 - 6 2 5 x 103; SC, 5 - 6 2 5 X 103). T h e s e c o n d f i n d i n g is t h a t t h e inc r e a s e in m i t o g e n i c r e s p o n s e w i t h i n c r e a s e in cell d o s e o f P B M C o r S C b e t w e e n 5 x 10 3 to 156 x 10 3 a p p e a r s linear. T h e t h i r d f i n d i n g is t h e s i m i l a r i t y in the m a g n i t u d e o f m i t o g e n i c r e s p o n s e b e t w e e n P B M C a n d SC at cell d o s e s r a n g i n g f r o m 5 x 10 3 to 156 x 10 3. T h e s e results w o u l d suggest t h a t the m i t o g e n i c r e s p o n s e o f P B M C in t h e p r e s e n c e of F L C a n d I L - 2 is c o m p a r a b l e to t h a t o f S C o v e r a w i d e r a n g e o f cell dose.

Correlation of the mitogenic response of PBMC and SC in presence of FLC and IL-2 T h e m i t o g e n i c r e s p o n s e of e q u a l n u m b e r s of P B M C a n d S C f r o m i n d i v i d u a l m i c e was t h e n a s s e s s e d in the p r e s e n c e o f F L C (1.25 x 1 0 6 / m l ) a n d I L - 2 (3.5 h a l f m a x i m a l u n i t s / m l ) . T h e results (Fig. 3) s h o w e d t h a t a r e m a r k a b l e d i r e c t l i n e a r c o r r e l a t i o n exists b e t w e e n the log o f t h e i r res p o n s e s o v e r a 1 0 0 0 - f o l d r a n g e of cell d o s e (i.e., 1 0 0 - 1 0 0 0 0 0 ) (slope = 1.054 + 0.037; a n i n t e r c e p t = 0.562; r = 0.989 + 0.003). T h i s d e m o n s t r a t e s

176

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Fig. 3. Correlation of the proliferative response to PHA of PBMC and SC from individual mice supplemented with an excess of FLC (1.25×106/ml) and IL-2 (3.48 half maximal units/ml). Each symbol represents the response of an individual mouse; number of cell doses used in this study, 6-8; number of mice, 6; the line represents the mean slope. The SEM of the slope is 0.037.

that a one-to-one relationship exists between the PHA-induced mitogenic response of PBMC and SC of individual mice when the cells are supplemented with F L C and IL-2.

Discussion In the past, attempts have been made to assess the immunological activities of PBMC of mice (Horneffer et al., 1976; Jarrett-Toth et al., 1979). However, we do not know of any study demonstrating in a quantitative manner the relationship between a response index of the PBMC and that of a major lymphoid organ, such as the spleen. The results presented here demonstrate for the first time that the proliferative activity of PBMC in response to P H A stimulation can be assessed in a quantitative manner over a wide range of cell doses (i.e., 2 × 103 to 156 x 103) by supplementing them with an excess of F L C (1.25 × 106/ml) and IL-2 (3.5 half maximal units/ml). The use of these two types of supplements was based on our previous study (Peterson, 1982), which showed that three cell types are required to induce a mitogenic response to P H A stimulation, namely the PHA-induced proliferating T cells, the IL-2 producing T cells, and accessory cells. This F L C + IL-2 combinational method of facilitation is

more sensitive than the method of using F L C alone (Peterson, 1982) or the method of culturing small numbers of cells in round-bottom wells (Horneffer et al., 1976). The results further showed that a one-to-one correlation exists between the mitogenic responses of PBMC and SC of individual mice over a 1000-fold cell dose range (i.e., 100-100000 per culture), as judged by the slope (1.054 + 0.037) and the coefficient of correlation (0.989 _+ 0.003) (Fig. 3). This correlation in mitogenic response is consistent with the one-to-one correlation in the percentage of T cells we observed in PBMC (33%) and SC (37%) (data not shown). This method of assessing the proliferative activity of PBMC in response to PHA in the presence of exogenous F L C and IL-2 creates a condition for assessing a single limiting proliferating cell type that incorporates [3H]thymidine. The same procedure can be used also to assess the activity of other types of limiting T cells involved in the P H A response. For example, supplementation of PBMC with exogenous FLC could provide the condition necessary for assessing IL-2 production by limiting T helper cells as was demonstrated in a previous study with SC (Peterson, 1984). This procedure should be applicable in assessing the activity of limiting T subsets in PBMC responsive to most T cell specific mitogens and antigens. The advantage of this technique is that it reduces the variation among individual small samples of PBMC. The quantitative method developed here should encourage investigators to assess in a time-dependent manner the effects of various exogenous and endogenous perturbants on the immune system of individual mice. This will eliminate the gnawing problem existing in all cross-sectional kinetic studies; i.e., the confounding effect on an immunologic index that can be caused by the variation existing between individuals sampled at different time intervals. Moreover, the method, when adapted for longitudinal study, should enable one to significantly reduce the sample size and therefore the animal budget.

Acknowledgements I thank Dr. T. Makinodan for helpful suggestions; Heidi Yellin and Joann Crawford for tech-

177 nical assistance; and the Department of Nuclear M e d i c i n e o f t h e U n i v e r s i t y o f C a l i f o r n i a at L o s A n g e l e s for use o f t h e i r 6 ° C o source.

References Casarett, A.P. (1968) Radiation Biology (Prentice-Hall, New Jersey) p. 90.

Homeffer, P.J. and Weksler, M.E. (1976) J. Immunol. Methods 11, 99. Jarrett-Loth, E., Segar, D. and Hong, R. (1979) J. Immunol. Methods 29, 201. Peterson, W.J. (1982) Cell. Immunol. 70, 260. Peterson, W.J. (1984) Fed. Proc. 43, 1688. Remington, R.D. and Schork, M.A. (1970) Statistics with Applications to the Biological and Health Sciences (Prentice-Hall, New Jersey) p. 260. Zeigler, J.B., Hamen, P.J, Dauier, W.W. and Penny, R.J. (1974) J. Immunol. 113, 2035.