CELLULAR IMhIUNOl~OGY

16,

237-250 (1975)

The Response of Human Peripheral Blood Lymphocytes to Determination of Cell Numbers Phytohemagglutinin:

Optimization and definition of conditions for studying lymphocyte function ill zitru resulted in exponential proliferation of lymphocytes from day 2 to day 5 with an average doubling time of 20 hr. The number of cells in culture on day 5 was S-10 times as great as the number initially planted and 10-20 times as great as the number surviving in culture on day -3. An improved pronase-cetrimide technique was used to tletermine the number of viable lymphocytes as a function of time after addition of PH:Z. The volume changes in nuclei, obtained after cetrimidc treatment, Lvere quantitated using a curve-fitting computer program. The response could be described in terms of an induction phase (O-Z days) characterized by a decrease in cellularity and an increase in nucIear volume, a proliferation phase (2-5 days) characterized by an exponential proliferation and a continued increase in the number of cells having a large nuclear volume, and a lysis phase (S-14 days) characterized by a decrease in cellularity and a decrease in nuclear volume. The results reported here suggest that the ratio of the number of cells cultured to the volume of culture medium was crucial for optimal transformation and proliferation, lo” cells/ml producing far better respones than lo” cells/ml.

INTRODUCTION In 1960 Nowell described the transformation and proliferation of human peripheral blood lymphocytes in response to phytolxmagglutinin (PHA) in vitro and showed that some lymphocytes were capable of mitosis and therefore were not end cells ( 1). Since then, the in vitro growth of lymphocvtes has become an _ important tool for studying lymphocyte functions ( 2, 3 j. The response of lymphocytes to PHA was initially evaluated by mitotic intlex or percent transformed cells because the agglutination of lymphocytes by PHA made hemocytometer counts both tedious and unreliable (2). Although these two methods quantitate the response at the time of measurement, the)- do not reflect the time course of changes in population size during the response. Radioactive thymidine incorporation into newly synthesized DNA by the proliferating population has been the method most often used, and its standardization (4, 5) has made the evaluation of the response more objective. Stewart and Ingram (6) reported a method for counting canine lymphocytes respondin g to PIrTA using a cetrimide counting solution that produced a monodispersed suspension of cell nuclei. These nuclei could be counted and the volume distribution could be obtained using an electronic particle counter.

Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

238

STEWART,

CRAMER

AND

STEWARD

The purpose of this report is to describe the PHA response of human peripheral blood lymphocytes in terms of cell number, cell morphology, and 3H-TdR incorporation as a function of time following stimulation with PHA, as assayed by an improved pronase-cetrimide counting technique. Using cell proliferation as a criterion, culture conditions were defined which significantly improved the response of lymphocytes to PHA. MATERIAL

AND METHODS

Culture Medium A modification of Eagle’s minimum essential medium, alpha-MEM (Flow Laboratories) was used (7, 8). The medium was supplemented with 100,000 units/l of penicillin, 100 mg/l of streptomycin, and 10% (v/v) fetal calf serum. The medium had a final osmolality of 290 * 10 mOsm, was sterilized by passage through a 0.22~pm filter, and was stored at 4°C. Preparation Medium Sodium bicarbonate and fetal calf serum were deleted from the medium used to separate lymphocytes from peripheral blood in order to avoid an alkaline pH shift. The pH was adjusted to 7.3 * 0.1 with 1 N NaOH, and the osmolality was adjusted to 290 * 10 mOsm by adding 1.5 g sodium chloride per liter of medium. Iron Particle Suspension One hundred milligrams of carbonyl iron (GAF Corporation, Type SF, special, New York, NY) were mixed with 100 mg gum arabic (Acacia), dry heat sterilized for 3 hr at 200°C (if autoclaved, it must be dried thoroughly to prevent caking and rusting), and suspended in 5 ml preparation medium at 37°C just prior to use. Red Cell Sedimentation Solution Ten grams of 150,000 dalton Dextran (Pharmacia Fine Chemicals, Piscataway, NJ) dissolved in 100 ml of phosphate-buffered saline (PBS : 0.20 g NaHsPOd* H20, 1.92 g Na2HP04, 7.80 g NaCl, 0.40 g KC1 dissolved in 1 liter HzO) was mixed with 100 ml of 3% (w/v) sod ium citrate in distilled water and 200 ml preparation medium. Phytohervtagglutinin One hundred milligrams of PHA-P (Difco batch No. 551099) were dissolved in 5.0 ml culture medium and stored at 4°C. Pronase Grade B pronase saline (0.15 M NaCl) Pronase was thawed enzyme was discarded

(Calbiochem, Los Angeles, CA) solution was prepared in at a concentration of 5 mg/ml and stored frozen (-20°C). and filtered through a 0.45-pm filter just prior to use. The after 2 hr at room temperature.

DETERMINATION

Counting

OF CELL

NUhlBIlR

239

Solutions

Red cells were counted in PBS. White blood cells were counted in a cetrimide solution (technical grade, Eastman Chemical, Rochester, IiU) , improved from the 30.0 g cetrimide, 0.372 g disodium EDTA, previous formulation (6), containing 8.268 g NaCl adjusted to a final volume of 1000 ml water (and the pH was adjusted to 5.0). The EDTA maintains the pH at 5.0, which is optimal for cytolysis, and chelates Mg’+ and Ca?+ ions to prevent aggregates.

Prejmration

of Lymphocyte Suspensions

Venous blood was drawn into heparinized glass tubes (BD Vacutainer NO. 4805 ) since the use of chelating agents as anticoagulants interferes with phagocytosis (9). One milliliter of iron particle suspension was immediately added to each tube and it was rotated horizontally at l-2 rpm for 1 hr at 37°C. The mixture was then diluted with 5 vol of preparation medium to enhance lymphocyte yield prior to centrifugation at 300 g for 10 min. The supernatant was discarded and the buffy coat and upper half of the red cell layer were mixed in a graduated cylinder with .5 ~1 of the red cell sedimentation solution. The bulk of red cells sedimented in 15-30 min. The lymphocyte-rich supernatant was removed, washed three times in preparation pellet was medium 1)) centrifugation at 300 g for 10 min, and the lymphocyte resuspended in culture medium. Red cell content was ascertained by counting in PBS at an aperture current of l/4. amplification of l/4, and a threshold of 40 usin g a Nuclear Chicago electronic particle counter. White cell content was ascertained by lysing the cells with cetrimide and counting the nuclei at the same settings used for erythrocytes. Viability was determined by incubating the cell suspensions for 10 min at 37°C with an equal volume of pronase before adding 10 ml cetrimide.

Lyl2pllocyt~~ Cultures Seven healthy donors of both sexes, ranging in age from 20 to 40 years old, were used in 21 separate experiments. Lymphocytes were mixed with PHA in 13 X IOOmm glass culture tubes (RTU Beckman No. 7816). sealed with micropore surgical tape (3-M Company, St. Paul, MN) and incubated upright and stationary at 37°C in a 9% CO9 humidified atmosphere.

Assays of Kcsponse Cell counts. Every 24 hr, replicate cell cultures were dispersed and incubated for 10 min at 37°C with an equal volume of pronase. The cell suspension was then transferred to 10 ml cetrimide using a Pasteur transfer pipette, and the culture tube was rinsed with cetrimide. Nuclei were counted using the electronic particle counter with multichannel analyzer at the settings mentioned above. n’uclear volume distribution. The multichannel analyzer accumulates pulses whose heights are approximately proportional to the volume of the nuclei which produced them. These pulses are sorted by height into 200 channels, generating a frequency distribution of nuclear volumes. The data were fit to the sum of two log normal distributions by a program which mathematically extracts two particle size tlistributions from a single composite distribution.

240

STEWART,CRAMERANDSTEWARD

Thymidine uptake. One-half milliliter of 3H-thymidine ( 3H-TdR, specific activity 6.0 Ci/mmole), made up to 3.0 ,&i/ml, was added to replicate cultures. The uptake was terminated 4 hr later with cold 5% (w/v) trichloroacetic acid (TCA). The precipitates were washed three times by centrifugation at 1500 g for 10 min with cold 5% (w/v) TCA. The precipitate was solubilized with hyamine hydroxide, transferred to liquid scintillation counting fluid, and assayed for radioactivity using a Packard liquid scintillation spectrometer. Cell morphology and autoradiography. Cultures were incubated for 18 hr with 0.1 &i/ml 3H-TdR. After incubation, each culture was washed twice with 5 ml of the preparation medium by centrifuging at 300 g for 10 min. The pellet was suspended in 0.05 ml human plasma and coverslip (22 X 22 mm) smears prepared. Some coverslips were mounted on glass slides and dipped in NTB-2 liquid nuclear track emulsion diluted 1 : 1 with distilled water. After 3 weeks of exposure, slides were developed in D-19 and stained with May Grunwald-Giemsa. Other coverslips were stained immediately with Wright’s stain to determine the percentage of transformed cells. Lymphocytes were considered transformed if they had fine nuclear chromatin, were enlarged, and had a distinct basophilic cytoplasm surrounding the nucleus. Blast cells were usually found in aggregates typical of PHA-cultured lymphocytes. RESULTS Isolation of Lymphocytes Relatively pure lymphocyte populations were desired to minimize cell debris created by dying polymorphonuclear leukocytes so cell counts would reflect changes in the lymphocyte population. After centrifuging the iron-laden phagocytic cells from the blood, the differential percentage of lymphocytes increased from 27% (range 1140) to 92% (range 70-100) with a recovery of 73% (52-88). After dextran sedimentation, the ratio of erythrocytes to lymphocytes was reduced from 2901 (range 1307-6060) to 134 (range 65-200). The recovery of lymphocytes after completion of these isolation steps which took 3-4 hr, was 53 % (31-71) and viability ranged from 85 to 100% in the final suspension as judged by either the pronase-cetrimide technique or by trypan blue exclusion. The final cell population contained small, medium, and large lymphocytes in varying proportions, as well as an unquantified number of aggregated platelets. PHA Dose Response The optimal dose of PHA was first determined by culturing lymphocytes for 5 days with final PHA concentrations of 4, 12, 36, 108, and 324 pg/ml and a final cell concentration of 1 X loj cells/ml. Six cultures were set up for each of the cell counts while duplicate cultures were used for the “H-TdR incorporation (except 12 pg/ml determination when six cultures were set up). Table 1 contains the results of this experiment. Using doubling time, obtained by assuming exponential growth from days 24, as an index of the lymphocyte response, a broad concentration range of PHA produced a similar stimulation of lymphocytes. However, when cell number is used as the index, the greatest response occurred using 12 pg PHA/ml. In this experiment, the doubling time ranged from 21 to 96 hr except for the highest PHA concentration for which the doubling time was sig-

DETERMINATION

OF CELL

TABLE

141

KUMRER

1

RESPONSE OF LYMPHWYTES TO DIFFERENT PHA CONCENTRATIONS Cells per culture

Day l’H.4 &g/ml) : .~ ___---0.2 1 2 .z 4 5 Doubling

time:

0

4

1.39f0.05" 1.48f0.03 1.41f0.08 1.53fO.l 1.7OztO.18 1.56xtO.08

1.27 fO.O1 0.024~0.1 1.11 f0.11 2.08 ho.03 4.80 ho.07 7.46 k0.78

(hr)b

22.7

12 1.16f0.02 1.14f0.15 0.98fO.l 2.02f0.05 6.71~0.56 8.43zt0.21 17.2 Incorporation

2 .z 4 5

751 6.59 182 540

17,910 44,554 64,944 49,153

27,183 52,850 78,916 42,529

(XlOP) 36

100

321

1.21 f0.06 1.22 zkO.03 0.963f0.04 1.32 fO.04 5.40 ho.14 6.28 ztO.02

1.16ztO.03 1.08f0.02 0.93f0.02 1.17dzO.04 2.63f0.31 5.45zkO.25

1.24 1tO.02 1.03 rtO.03 0.895-fO.l 1.06 ztO.03 1.78 zkO.03 2.29 fO.20

19.,z

31.4

48..1

25,962 62,042 60,710 71,758

20,469 33,695 43,769 78,593

of 3H-TdR 26,334 86,230 66,505 52,416

(dpm)

‘I Standard deviation. ‘>Calculated between day 2 and day 4 using the equation N = N2t’7’d, where N is the number of cells at time t (days), No is the number of cells present at the beginning of the proliferation phase (day 2) and Td is the doubling time.

nificantly increased. Microscopic examination of nuclear suspensions showed some unlysed agglutinated cells at the highest PHA concentration, but not at the lower concentrations. The incorporation of 3H-TdR, also shown in Table 1, indicates extensive cell proliferation in all cultures stimulated with PHA. Kinetics of the Response The kinetics of the PHA response were measured in more detail using 12 pg PHA/ml and an initial cell concentration of 1 X lo5 lymphocytes/ml. Figure 1 shows the kinetics of the change in cell number of PHA-stimulated lymphocytes obtained from seven different individuals in 21 separate experiments. The PHA response could be described in terms of a 2-day induction phase, characterized by an average 4370 decrease in a cell number with a nadir usually on day 2, a proliferative plznse characterized by a rapid increase in cell number for days 2 through 5, and a 1ysi.s phase, occurring after day 6, characterized by loss of viability and a gradual decrease in cell number. The slope of the proliferative phase, determined by the method of least squares using the data of clays 2, 3, and 4, indicates a population doubling time of 20 hr. The mean increase in cell number was .5.7-fold (range 4- to IO-fold) over the number initially cultured, and lo- to 20-fold over the number surviving on day 2. In three of the experiments described above, incorporation of SH-TdR was measured, and these data are shown in Fig. 2. The rate of 3H-TdR incorporation with time increases up to day 4, substantiating the proliferative response induced by PHA as measured by cell counting.

242

STEWART,

CRAMER

AND

STEWARD

%-eeehY

0

14

DAYS IN CULTURE FIG. 1. Proliferation of PHA-stimulated human lymphocytes. The percentage of cells remaining in culture as a function of time is shown for 21 experiments utilizing blood from seven individuals. Each point represents the mean of 2-4 cultures for each of the experiments using 10’ cells/ml and 12 pg PHA/ml.

Figure 3 shows the cell survival of the unstimulated lymphocyte controls. We routinely observed, in individual experiments, an increase in cells in control cultures between days 5 and 7. We ascribe this increase to the antigenic stimulation of a small subpopulation of lymphocytes by the presence of fetal calf serum in the culture medium. Donors had no history of allergy to penicillin or streptomycin.

I ‘#O

A

I 2

1 3

4

f 5

,

DAYS IN CULTURE

2. Incorporation of *H-TdR into newly formed DNA by PHA-stimulated human lymphocytes. An initial cell suspension of 106 lymphocytes was stimulated with 12 pg PHA in 1.0 ml cultures. Cell cultures were pulsed for 4 hr at 2.0 &i *H-TdR/ml and the cells assayed for incorporation. The symbols represent individual cultures from three separate experiments. FIG.

DETERMINATION

“0

1

OF CELL

2

4

243

NUMBER

6 6 10 OAK? IN CULTURE

d

12

14

FIG. 3. Survival of human lymphocytes ila zsitvo. The percentage of cells surviving in cultures without PHA as a function of time is shown. Each point represents the mean of Z-4 cultures in several experiments.

Nuclea,r Volume Distributions We previously reported an increase in nuclear volume of lymphocytes following their transformation by PHA (6). This increase can be quantitated from the distribution of pulse heights produced in the particle counter by the nuclei and accumulated by a multichannel analyser. In Fig. 4 graphic output of the computer analysis of the nuclear volume frequency distribution is presented. The two distributions derived from the data and referred to as the nontransformed and the transformed lymphocyte nuclei are identified in

C

22

57

92

127

162 22

57

CUBIC

92

1.27

162 22

57

92

Q7

162

MiCRONS

FIG. 4. Volume frequency distributions of human lymphocyte nuclei. The relative number of lymphocyte nuclei is plotted as a function of their volume. In each distribution, the nuclei from the nontransformed and transformed lymphocyte populations have been separated by computer analysis. In (E) and (F) only a single population could be fit. The distribution of lymphocyte nuclei is shown in (A) for cells cultured without PHA after 2 hr of culture, in (B) for lymphocytes 1 day after PHA stimulation, in (C) 2 days after, in (I)) 3 days after, in (E) 5 days after, and in (F) 8 days after PHA stimulation.

244

STEWART,

CRAMER

AND

STEWARD

Fig. 4A. The number of nuclei in each population was calculated by the computer to give the percent transformed cells shown in Table 2. Figure 4A obtained at 2 hr shows the control lymphocytes to which PHA was not added. Two populations of cells are shown; the major component is composed of nuclei derived from small nontransformed lymphocytes. This figure is representative of the distributions of the control cell nuclei throughout the culture periods. A slight increase in the number of transformed cells was observed in the controls on days 5, 6, and 7. By day 1 after addition of PHA (Fig. 4B) there was an increase in the proportion of nuclei in the transformed population (Table 2). The dynamics of these changes are shown in Figs. 4B4E, representing days 1, 2, 3 and 5, respectively. The distribution of nuclei obtained on day 4 was identical to that shown for day 5 in Fig. 4E. Beginning on day 6, there was a shift in nuclear volume to an intermediate volume between the untransformed control nuclei and the stimulated transformed nuclei. This shift was complete by day 8 as shown in Fig. 4F. Transformed Cells in Cycle The number of cells in cell cycle was determined as well as the percent of transformed cells measured morphologically. Since the slope of the proliferation curve (Fig. 1) is consistent with a doubling time of 20 hr, PHA-stimulated and control lymphocyte cultures were incubated with 0.1 &i 3H-TdR for 18 hr. With this incubation time nearly every cell in cell cycle should be labeled. The data comparing the percent transformed cells as determined by morphological criteria and the computer calculation shown in Table 2 correlate well. The absolute number of cells in cycle shown in Table 3 was determined by multiplying the percent labeled cells by the number of cells per culture. Not until day 4 were nearly all the cells in cycle. These data suggest that after addition of PHA transformed lymphocytes are continuously recruited into cycle from days O-3. TABLE

2

LYMPHOCYTETRANSFORMATIONDETERMINED BYNUCLEARVOLUME LABELINGAND MORPHOLOGY Day

Cells per culturea

Fraction Nuclear volume6

transformed Morphology

Labeling indexc

(%I 0 1 2 3 4 5 6

100,000 57,000 68,000 140,000 355,000 570,000 570,000

= dt

l?(t) + $ 2(“‘)R(L

-

12i),

(1)

i=l

where the first term on the right, R(t), is the number of noncycling cells at time t recruited into cycle per hour, and subsequent terms give the rate that cells are added to the cycling compartment by the division of previously recruited cells.

DETERMINATION

OF CELL

NUMRER

249

Since presumably no cells were recruited for t < 0. n is the largest integer < t/12. Gi\-en the two additional assumptions that (iv ) no cells are in cycle at time t = 0, and (v) the recruitment rate (number of noncycling cells recruited into cycle per hour) is a step-wise continuous function, the solution to Eq. 1 is t R(f) dt’ + 2 2’1-1, ’ ‘I” X(t7 df’. (2) i 0 10 t=1 where the first term is the number of cells recruited into cycle and subsequent terms are the number of cycling cells added through mitotic activity. l?or simplicity, we now assume that the recruitment rate, Ii(t), is a constant over each 23-hr interval; i.e., R(t) = RI for 0 < t < 24, R(t) = X2 for 2-C< t < 48, etc. Equation 2 now becomes N(t)

=

k-(i/2)--a,

Nk = 24(;

Rj + F 2’i-1’[ j=l

i=l

x

Rj + ~iR/r-(i-l)/2j}r

(3)

j=1

where NI, is the number of cells in cycle at the end of the kth 24-hr interval and (Y~assumes the value 0 when i is even and 3 when i is odd. By expanding the second summation of Eq. 3 and combining coefficients of RI, IZz. Xi

The response of human peripheral blood lymphocytes to phytohemagglutinin: determination of cell numbers.

CELLULAR IMhIUNOl~OGY 16, 237-250 (1975) The Response of Human Peripheral Blood Lymphocytes to Determination of Cell Numbers Phytohemagglutinin: O...
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