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:

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


0.32 0.72 0.89 0.99 0.99 0.78

0.02 0.17 0.64 0.95 0.84 0.64

d From least-squares line of Fig. 1. b Fraction transformed nuclei = number of nuclei in second population/total cells. 6 Labeling index = labeled cells counted/total cells counted. d Nuclei beginning to revert to an intermediate size between that of the transformed small lymphocytes.

cells and

DETERMINATION

OF CELL

TABLE RECRUITMENT

Time interval

AND PROLIFERATION

k

o-24 24-48 48-72 72-96

1 2 .z 4

3 OF PHA-STIMULATED

1,428 13,192 90,300 339,025

5,928 52,768 361,025 Total

LYMPHOCYTES

Recruited

Without recruitment*

Cells in cyclea (Nk)

hrs

KINETICS

24.5

NUMBER

cells in interval

Ratec i&)

Number ___--.-..

41.2 201.8 1,042.57 -615.91

987 4,843 25,021 None

cells recruited

Progeny” 40.5 2,421 12,511

= 30,700

a Total cells in culture multiplied by the labeling index (from Table 2). * The number of cells which would exist at the end of the present time interval if there was no further recruitment and all cells in cycle at the beginning of the interval divided with a generation time of 12 hr. c Calculated using Eq. 5 of the Appendix. d The progeny, accumulated at the end of the interval, of cells which were recruited within the interval.

Response of 106 Lymphocytes Usually, lymphocyte responses to PHA are obtained using lo6 or more lymphocytes per 1.0-2.0 ml culture. Our results are shown in T;ig. 5 for the response of 10” lymphocytes in 1.0 ml culture medium along with the response measured for an initial concentration of IO5 lymphocytes/ml. Clearly IO6 cells/ml do not respond to PHA as well as those cultured at lo5 cells/ml.

I

2

3

4

8-L

5

i 6

DAYS IN CULTURE FIG. 5. Comparison of the lymphocyte response at lo” and 1OP cells/culture. Lymphocytes (I@‘) were cultured with 12 fig PHA or with no PHA in 1.0 ml. The mean of replicate cultures from two experiments is shown. Also shown for comparision is the proliferative response of 10’ lymphocytes repeated from Fig. 1.

246

STEWART,

CRAMER

AND

STEWARD

DISCUSSION Although population kinetics during the proliferative response to PHA has been previously investigated, cell number has not been extensively studied throughout the course of the response (10-12). Using this parameter, we observed an exponential increase in cellularity of PHA-stimulated human lymphocytes; the slope of the growth curve gives a population doubling time of 20 hr (Fig. 1) . The term doubling time has been used, alternatively, to define the time it takes for the rate of DNA synthesis to double. Thus, McIntyre (13) found a doubling time in PHA-stimulated human lymphocytes ranging from 26 to 31 hr. Valentine (14) reported a much shorter doubling time of 12 hr and interpreted this as representative of the generation time, since cultures of twice as many celIs had twice the rate of DNA synthesis. It may not be valid, however, to use DNA synthesis doubling time to calculate the generation time since the number of cells synthesizing DNA at any time is the sum of those originally stimulated and presumably continuously dividing plus those newly recruited. Thus the rate of DNA synthesis would be accelerated by recruitment and would tend to underestimate the generation time. Only when there is no recruitment would such an analysis be expected to yield the generation time. Lohrmann et al. (15) used PHA to stimulate canine lymphocytes and hydroxyurea (for 14 hr) to synchronize the population beginning on day 3. Their SH-TdR incorporation curve suggested good synchrony with little or no recruitment, and they found a generation time of 14 hr. Our data (discussed below) are in agreement with the conclusion that recruitment is negligible after day 3, although our maximum recruitment rate was between 48 and 72 hr rather than between 30 and 53 hr of culture as reported by Lohrmann et al. (1.5). Our own SH-TdR incorporation curve (Fig. 2) shows an initial DNA synthesis doubling time of 12 hr (but the rate continuously changes) and becomes longer with time. Although this supports the lower figure of Valentine, McIntyre’s value is consistent with the time lapse cinematographic study by Marshall et al. (16) of two PHA-stimulated human lymphocytes which exhibited cycle times of 23 and 38 hr. However, their later and more extensive study using pokeweed mitogenand antigen-stimulated human lymphocytes documented a wide range of generation time from 7 to over 24 hr, with most values in the range of 8-12 hr (17). The earlier results with PHA were then interpreted as having been obtained under suboptimal culture conditions. Generation time for PHA- stimulated human lymphocytes was estimated by Sasaki and Norman (18) at 22 hr using the percent labeled mitoses method. Wilson (19) used the same technique and obtained a value of 19.5 hr which contrasted with his finding of a generation time of only 15.1 hr for allogeneically stimulated human lymphocytes. Bach (20) found a generation time of 18-21 hr for allegeneically stimulated human lymphocytes. It should be pointed out that nutrient utilization requirements for human lymphocytes in tissue culture are not well defined at present. Such requirements have been shown to be a significant factor in cultures of mouse peritoneal exudate cells (8) and are currently being studied in our laboratory for PHA-stimulated human lymphocytes. We believe that variability in culture conditions is most likely responsible for the above cited controversy in the literature. The use of different stimuli also contributes to the confusion. Since antigen, allogeneic cells, and pokeweed mitogen may stimulate different subpopulations of lymphocytes than does phytohemagglutinin, it may not be valid to assume similar generation times for

DETERMINATIOi’G

OF

CELL

NUMBER

247

each subpopulation. Until definition of lymphocyte requirements ifi &ho allows optimization of culture conditions, it seems reasonable to take the position that, at present, the generation time for PHA-stimulated human lymphocytes under optimal culture conditions is not known. The magnitude of the cell population in the proliferative phase (40-120 hr) is a function of the cell proliferation rate, the recruitment rate of cells into cycle, the rate of cells leaving cycle, and the cell death rate. It is possible to estimate the recruitment rate using our data if certain assumptions are made. The data are treated as described in the Appendix and the results are shoed in Table 3. Tn order to maximize the role of clonal proliferation in pro(lucing the observed growth. a 12-hr average generation time with no cell death occurring between 24 and 96 hr was assumed. This minimizes the calculated recruitment required to account for the total cell number in culture. From Table 3, it can be determined that the average recruitment rate (column 5) increases exponentially in each 24-hr interval through tlav 3. The cells recrnitecl during each 23-hr interval produce some progenv lvithin that interval. These progeny, shown in the last column of the table, also contribute to the number of cells in cycle at the end of the interval. In column 4 is given the number of cells that would be expected at the end of the interval if there \vere no recruitment within the interval. Clearly. the observed cellularity of the cultures through day 3 can l)e accounted for only if there is continuing recruitment. In the interval 72-96 hr. the recruitment rate falls to a negative value. We have interpretetl this to mean that recruitment stops after day 3. Cells must begin to leave cycle after this point since more cells could have been produced bv the cells in cvcle on day 3 than were actually observed on day 4. Our analysis has shown that as little as 3070 of the original population could have responded to PHA. Tllis fraction would be greater if cell death occurs during 24-96 hr, if cells leave cycle before 72 hr, or if cells have a longer generation time than we assumed. During the induction phase (O-48 hr), a decrease in the number of cells per culture was observed. Aggregation of nuclei commonly occurs if the pH of the nuclear suspension is above 5.5, so we maintained the pFI below this by adding tlisodium EDTA to the cetrimide solution. The presence of some aggregated nuclei and some incompletely lysed cell aggregates was insufficient to account for the observed decrease in counts. Since only 15-200/O of the I\-mphocytes from human peripheral blood are B lymphocytes (21) and since we often observe a 40-60s loss of cells during the induction period, it seems unlikely that the death of B lymphocytes alone could account for this cell 10s~. PIHA toxicity to some lymphocytes might be responsible ; however, the lack of ;i significant dose dependent toxicity (Table 1) would argue against this possibility. The drop in cell number during this phase may be due to the failure of some I!-mphocytes to recover from the insults associated with transfer to in vifro conditions, while control Iympllocytes are permitted such recovery because they are not stimulated. Cell death again becomes an increasingly significant component of the kinetics after day 4 (lysis phase) as the number of cells per culture plateaus ant1 then decreases. even though continued incorporation of 3H-‘l‘dR suggests that cell proliferation is still occurring. An increase in the tleath rate c\-ould be expectetl as culture conditions become poor due to the depletion of cssentia] nutrients antI Ihe accumulation of waste products and Iymphokines.

248

STEWART,

CRAMER

AND

STEWARD

In this study the lymphocyte fraction was obtained using the carbonyl iron method (6) for removal of phagocytic leukocytes and dextran sedimentation to reduce erythrocyte contamination. In preliminary experiments using FicollHypaque separated lymphocytes from whole blood, we have obtained similar kinetics in response to PHA stimulation. These preliminary results suggest that different methods for separation do not influence the result. We attribute the poor response of lo6 cells/ml to nutrient depletion. From studies on the nutrient depletion of peritoneal exudate cells (S), it seems likely that some essential components may be depleted after day 3 when higher cell concentrations are used, resulting in the death of cells which might otherwise continue to proliferate. The prevalent viewpoint is to consider PHA as a nonspecific stimulus of lymphocyte proliferation, in analogy to the stimulation of small subpopulations of lymphocytes by specific antigens. This concept does not account for the significant decrease in cell number which we observed during the induction phase and during the lysis phase. Although we have offered an alternative explanation for the decrease in cell number in the inductive and lytic phases, it is attractive to speculate that these observations may reflect the functional response of the responding lymphocyte population to PHA. It is known that cell death is generated by interaction of antigen-specific cytotoxic lymphocytes with their target antigen (22)) and it has long been known (23) that PHA generates cytotoxic lymphocytes. Since PHA probably interacts with lymphocytes of different antigenic specificities, the cell death caused by PHA may be analogous to that generated by interaction of antigen specific cytotoxic lymphocytes with their target antigen. Extending the analogy, PHA induces the clonogenic precursor cells of varying specificities to proliferate as if it were their specific antigen and generates new cytotoxic lymphocytes which in turn react with PHA in the culture and die. ACKNOWLEDGMENTS The authors wish to thank the members of the Se&on of Cancer Biology and Dr. Stuart Kornfeld for their comments on this report. This work was supported by USPHS Grant No. lPOZCA13053 from the NCI. APPENDIX The analytical method for determining the rate of recruiting transformed lymphocytes into cycle incorporates the following assumptions. (i) The intermitotic period of every cycling cell is 12 hr (17). (ii) C eI1s are initially recruited from resting phase into early G1 phase (24) ; they divide 12 hr later. (iii) There is no cell death from the cycling compartment, . each mitosis results in one additional cycling cell. With these three assumptions, the number of cycling cells at time t, N(t) , is given by the equation

dN (t> = 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<, etc., it is found that Eq. 3 can be written ~~ = 24 f: &. + 24 i f=l j=1

(3 X 2gk-2i--I - 1)Ri

(4)

or

jJlk = 36 C 2z(k--j)Rj. j=l

( .i 1

Jn Eq. 4, the first summation yields the total number of cells recruited into cycle from the resting phase and the second summation the total number of cycling cells accumulated by the end of the kth 24-hr interval through mitotic activity of those recruited. The jth term of the first summation is the number of cells recruited during the jth 24-hr interval while the jth term of the second summation is the number of cycling cells accumulated by the end of the kth 24-hr interval as a result of the mitotic activity of cells recruited during the jth 24-hr interval. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. Y.

Nowell, P. C., Cancer Res. 20, 462, 1960. Ling, N. R., In “Lymphocyte Stimulation,” North-Holland, Amsterdam, 1968. Naspitz, Ch. K., and Richter, hf., Pragr. Allergy 12, 1, 1968. Schellekens, P. Th. A., and Eijsvoogel, V. P., Clitz. lZ.rfi. I~~r~?~zof. 3, 571, 1968. Fitzgerald, M. G., Cl&. E.rP. Iwmnol. 8, 421, 1971. Stewart, C. C., and Ingram, M., Blood 29, 628, 1967. Stanner, C. P., Eliceiri, G. L., and Green, H., n’aturc SCW Biol. 230, 52, 1971. Stewart, C. C., J. Retiruloendothcl. Sot. 14, 332, 1973. Dubos, R., and Hirsch, J. G., Ipt “Bacterial and Mycotic Infections of Man,” 4th etl., p. 225. Lippincott, Philadelphia, PA, 1965. 10. Eurenius, K., and McIntyre, 0. R., Int. Arch. Allcr,qv 37, 393, 1970. 11. Tsutsui, I., Acta Haematol. Japan 30, 865, 1967. 12. Tsutsui, I., Acta Haematol. Japan 30, 884, 1967.

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13. McIntyre, 0. R., and Ebaugh, F. G., Blood 19, 443, 1962. 14. Valentine, F. T., In “Cell-Mediated Immunity 1% Y&o Correlates,” p. 14. University Park Press, Baltimore, MD, 1971. 15. Lohrmann, H., Graw, C. M., and Graw, R. G., J. Exp. Med. 139, 1037, 1974. 16. Marshall, W. H., and Roberts, K. B., Quart J. Exp. Pkys. 50, 361, 1965. 17. Marshall, W. H., Valentine, F. T., and Lawrence, H. S, J. Exp. Med. 130, 327, 1969. 18. Sasaki, M. S., and Norman, A., Nature (Loadon) 210, 913, 1966. 19. Wilson, D. B., Blyth, J. L., and Nowell, P. C., J. Exp. Med. 128, 1157, 1968. 20. Bach, F. H., Bock, H., Graupner, K., Day, E., and Klostermann, H., Proc. Nat. Acad. Sci. U.S.A. 62, 377, 1969. 21. Rabillion, E., Colon, S., Grey, H., and Vnarue, R. R., J. Exp. Med. 133, 156, 1971. 22. Henney, C. S., J. Immunol. 107, 1558, 1971. 23. Holm, G., Perlmann, P., and Werner, B., Nature (London) 203, 841, 1964. 24. Bender, M. A., and Prescott, D. M., Exp. Cell Res. 27, 221, 1962.