Proliferative Capacity and Differential Mitogen Sensitivity of Various Precursors of B Lymphocytes Analysed by Secondary Culture of Single Clones

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 5, pp. 501-512, 1981. 0145-305X/81/030501-12$02.00/0 Printed in the USA. Copyright (c) Pergamon Press Ltd. All rights reserved.

PROLIFERATIVE CAPACITY AND DIFFERENTIAL MITOGEN SENSITIVITY OF VARIOUS PRECURSORS OF B LYMPHOCYTES ANALYSED BY SECONDARY CULTURE OF SINGLE CLONES 1

L. Lafleur 2 and G. Mercier Institut Armand-Frappier, C.P. 100 Laval-des-Rapides, Qu~bec, Canada H7N 4Z3 and Division of Experimental Medicine, McGill University

ABSTRACT Mouse bone marrow B lymphocyte precursors were fractionated by sedimentation velocity and cultured for various time intervals with 2 mitogens added sequentially or together. The distribution of the number of mature lymphocytes was measured and compared with the distribution of precursor cells at various stages of differentiation. It Was found that the detection of late-responding precursors was highly dependent on stimulation with 2 mitogens, LPS and agar extract. These precursors could be separated into a population of small homogeneous cells and one of large heterogeneous cells. The capacity of these precursors to differentiate to B lymphocytes and their selective mitogen sensitivity was analysed in secondary cultures initiated from primary mass cultures or from limiting-dilution cultures of precursor cells. The results suggest that, after long culture times, larger cells have a higher capacity to produce new mitogensensitive cells.

INTRODUCTION . . In adult animals, B lymphocytes are continually regenerated through the differentiation of pluripotent stem cells (1, 2). Various functional properties of the cells change as they differentiate and much still remains to be learned about the phenomenon. For e~ample, it will be important to better understand the process of the acquisition of a given antibody specificity (3), the mechanisms regulating the clone size of individual ~pecificities or determining the temporary susceptibility of lymphocytes to tolerance Induction (4). An investigation of these events will depend on a complete description of the stages of differentiation of progenitor cells into B lymphocytes and on functional assays for each of these populations. Efficient culture techniques have been developed OVer the last few year (5) and mitogens have been found to stimulate populations of

n 2)

Supported by the Medical Research Council of Canada "Chercheur-Boursier" of the Conseil de Recherche en Sant~ du Qu~bec 501

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precursor cells (6). In a previous paper, a number of populations of B lymphocyte precursors differentiating in adult bone marrow were described (7). In the present paper, an analysis was made of the kinetics of differentiation, of the proliferative capacity (clone size) of individual progenitors of B lymphocytes in the course of differentiation and of the influence of the cumulative addition of different mitogens on these parameters.

MATERIALS AND METHODS Animals: BALB/c mice, 8-12 week old specific-pathogen-free males or females were bought from Biobreeding Farms (Ottawa, Ontario) and kept in filtered cages. SpragueDawley rats 50-200 gr, males or females were obtained from Canadian Breeding Laboratories (St-Constant, Quebec). Cell suspensions: Mouse bone marrow cell suspensions were made by flushing femurs with Earle's balanced salt solution (EBSS) and washing the cells twice with RPMI-1640 medium. Rat thymus cell suspensions were prepared by teasing the organ with forceps in EBSS and washing twice in RPMI-1640. Except for size separation of the bone marrow cells, all manipulations were carried out at room temperature. Cell separation: Bone marrow cell suspensions were separated by velocity sedimentation aC90rding to Miller and Phillips (8) with minor modifications. Briefly, cells at 1.5 x 10 Iml were loaded in 20 or 40 ml of 0.1% Ficoll (Pharmacia, Montreal) in EBSS over a gradient of 0.2% to 1.0% Ficoll. Sedimentation time was 3 hours at 6°C, 15 ml fractions were taken, counted with a Coulter counter (Hyaleah, Flo) and usually pooled in groups of 2. Following culture, total activity per fraction was generally reported in the Results rather than enrichment profiles as these were not thought as meaningful for the purpose of the experiments. Experimental protocols: The aim of the experiments was to analyse the differentiation in vitro of precursor cells into mature B lymphocytes. As shown in Figure 1 bone marrow

Primary cultures A-

Mass 4 (lx10 -lx10 6/ml)

Secondary cultures (limiting-dilution) 1)

2)

Mitogen~

5-6 days

IgM-PFC's

---------

2-11 days with ____, _______ / mitogens

Mitoge3>IgM-negative 6 days supernatants

B- Limiting-dilution (10-20 cells/cult)

2) -------1)

Mitogens:> 5-6 days FIG. 1

Experimental protocols

IgM-negative supernatants

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cells were fractionated a~d cultured followin61 either of 2 general protocols, "massculture" conditions (1 x 10 cells/ml to 1 x 10 cells/mI) or limiting-dilution conditions (10-30 cells/culture). First, these cultures were stimulated with a water-soluble agar extract (AE) (9) and/or lipopolysaccharide (LPS) and the response of the mature lymphocytes estimated after 5-7 days, IgM-secreting cells for mass cultures, IgMnegative culture supernatants for limiting-dilution cultures. To measure at various times the production in the cultures of new lymphocytes, secondary limiting-dilution cultures were set up from pools of mass cultures or from individual limiting-dilution cultures. These were restimulated with mitogens, cultured for a further 6 days and the supernatants analysed for the presence or absence of IgM. Minor modifications of this general protocol are indicated where appropriate. Cultures: Culture medium 5was RPMI-1640 supplemented with 15% fetal calf serum ZFlow Laboratories), 2 x 10- M 2-mercaptoethanol, penicillin and streptomycin. Bone marrow cell fractions at the appropriate concentration were mixed with an equal volume of rat thymus cells at 4 x 10 6/ml (10), mitogens were added (AE 50 ug/ml and/or LPS, 40 ug/ml, final concentrations) and dispensed in Linbro plates in triplicate 0.1 ml cultures or two 60-well Terasaki plates (Falcon) in 0.01 ml volumes with Hamilton syringes (ll). The cultures were gassed with a mixture of 83% N 2, 7% 0,. and 10% CO in humidified boxes 2 and incubated at 37°C for various periods of time as inClicated in the R~sults section. For secondary cultures of cells cultured initially at 1 x 10 /ml, pools of 3-5 cultures were scrape?7 from the wells with a rubber policeman, washed twice in the presence of 4 - 7 x 10 thymus cells and readjusted to 3,000 cells/ml calculated from the number of cells seeded in the primary cultures. Mitogens were added and the cells distributed in 120 wells in 0.01 ml volumes for limiting-dilution analysis. For secondary cultures of cells cultured initially at or around limiting dilution, individual 0.01 ml cultures were aspirated up and down many times with a ~icropipet and transferred directly to 1 ml of a suspension of rat thymus cells at 2 x 10 /ml. Mitogens were added as indicated and the mixture of cells redistributed in :30 or 60 microwells. These cultures Were incubated for a further 6 days under the conditions described above. Detection of 19M synthesis: Individual IgM-secreting cells were scored as plaque-forming cells (12) using sheep red blood cells coated with rabbit anti-MOPC-I04E by the CrCl 1 method of Molinaro et al. (13). The antiserum was monospecific for IgM as determinea by immunoelectrophoresis. The presence or absence of IgM in the supernatant of the ~ultures was e~imated directly by adding sequentially to the microwells at 1 hour intervals 5 x 10 anti-IgM-sensitized red cells, an appropriate dilution of rabbit anti-Ig serum and complement, all in 0.001 ml volumes. The frequency of mitogen-sensitive cells was estimated by limiting-dilution cultures .on the basis of the percentage of cultures negative when tested for the presence of IgM in the supernatant of the cultures. The frequency and/or the total number of lymphocytes stimulated was then calculated by multiplying by the number of cells cultured and the number of cells per fraction when necessary.

RESULTS After size separation of bone marrow cells, most cells responding early to polyclonal activators were recovered in the small-lymphocyte region of the cell distribution while if cells were cultured for longer time periods a response began to appear in fractions of larger cells (7, 14). To analyse in more details the cellular origin of the late response, bone marrow cells were fractionated according to size and cultured as shown in protocols AI, A2, Bl in Figure 1. The initial number of LPS-sensitive cells present 'in each fraction was determined by 5 or 7 day cultures at limiting dilution. The number of IgM-PFC's was also measured after 7 days continuous culture. Simultaneously, each fraction was precultured for 2 days at 1 x 10 /ml in the presence of LPS or LPS plus AE. The number of LPS-sensitive cells present after these 2 days of stimulation was

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TABLE 1 Lymphocyte Production by Bone Marrow Fractions during 2 days in mass cultures with AE and/or LPS

Sedimentation velocity (mm/h)

Response X 10- 3 in secondary culturesc )

Response X 10- 3 in primary cultures 5 daysa) LPS

AE

7 daysa) LPS

+

AE

PFC'sb) LPS

+

LPS

AE

LPS

+

LPS

AE +

LPS

LPS

3

816

1,380

956

2,110

10,200

38,000

3,920

11,100

4

299

730

766

1,180

12,500

15,400

2,850

6,200

5

97

230

130

454

690

6,700

610

880

6

67

89

149

149

820

3,900

399

410

7

38

18

84

77

420

1,400

136

410

a)

Initial number of mitogen-sensitive lymphocytes/fraction estimated by 5 day or 7 day limiting-dilution cultures.

b)

IgM-secreting cells/fraction measured after 7 days of culture at 1 x 10 4 cells/mi.

c)

Primary cultures for 2 days at 1 x 10 4 cells/ml and secondary cultures at limiting dilution for 5 days.

estimated by secondary cultures at limiting dilution. As already shown (7), one can see in Table 1, more lymphocytes were detected after 7 days of culture at limiting dilution than after 5 days and the relative increase was more pronounced for fractions of larger cells: the number of responding cells found in the 3 and 5 mm/h fractions was increased by only 20% while it more than doubled in the other fractions. The measurement after 2 days of primary culture of the number of lymphocytes capable of enough proliferation and secretion to yield positive cultures shows that cells of all the fractions did not proliferate and differentiate to the same extent. There was a large increase in the number of lymphocytes between days 0 and 2, specially at 4 mm/h with LPS and at 3 mm/h with AE. A quantitative analysis of the results based on 3 different and not mutually exclusive hypotheses is shown in Table 2. Assuming in the first one that all the lymphocytes detected after a period of preculture of 2 days came only from lymphocytes that were initially present and that were therefore detected after 5 days in conventional cultures, the ratio of the number after 2 days over the initial number measures the extent of proliferation during those 2 days (columns 1 and 2, Table 2). If in a second hypothesis some mature cells respond slowly and only the day 7 of continuous culture measures the initial number of mature lymphocytes, the extent of proliferation for the various fractions is shown in columns 3 and 4, Table 2. Another hypothesis is that essentially all the lymphocytes stimulated initially lost after 2 days the capacity to proliferate enough to yield positive cultures and that only lymphocytes newly formed during the 2 days of preculture possessed such a capacity. Under those conditions, the

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TABLE 2 Ratio of the Number of Lymphocytes present in Culture after 2 days over the number of Lymphocytes newly formed or over the number initially present as measured after 5 days or 7 days of culture Velocity

day 5 a) measurement

day 7 b) measurement LPS d)

LPS d)

AE e)

newly-formed c)

LPS d)

+ LPS

3 4 5 6 7

4.8 9.5 6.3 5.9 3.6

8.1 8.5 3.8

4.1 3.7 4.7

5.3 5.3 1.9

4.6

2.7

22.8

1.6

27.8

15.3 13.8

2.7

6.1 18.5 4.9

5.3

3.0

6.9

3.9

6.8

a)

Ratio of the number of lymphocytes measured in Table I after secondary culture over the day 5 number of lymphocytes.

b)

Ratio of the number of lymphocytes measured in Table 1 after secondary culture over the day 7 number of lymphocytes.

c)

Ratio of the number of lymphocytes measured in Table 1 after secondary culture over the difference between day 7 and day 5 lymphocytes.

d)

Primary and secondary cultures stimulated with LPS.

e)

Primary and secondary cultures stimulated with a mixture of LPS and AE. ratio of the number measured after secondary culture over the di ffe,rence between days 7 and 5 would be an approximation of the number of lymphocytes per newly formed clone (columns 5 and 6). It appears from the last 4 columns that if mitogen-sensitive cells present in the cultures after 2 days were derived from mature lymphocytes initially present, with one exception they had had time for 1 to 3 divisions, depending on the fraction or the time of mitogenic stimulation. However, there is no consistent relationship between the initial number of lymphocytes and the one measured after 2 days. As expected, the last type of analysis yielded much higher values and clone size ranges from 3.0 to 24.5 lymphocytes, i.e. 2 to almost 5 divisions. It also appeared from these'results that whatever the type of analysis, stimulation with a mixture of mitogens did not systematically result in larger clones and fractions of smaller cells still produced relatively more lymphocytes. A higher resolution comparison of the initial number of LPS-sensitive lymphocytes with tge number of lymphocytes recovered after a period of primary culture of 5 days at 1 x 10 Iml with LPS is shown in Figure 2. The secondary Ig-PFC's induced by LPS from the lymphocytes recovered after the primary culture period were also measured in this experiment. The initial number of LPS-sensitive lymphocytes determined by limitingdilution analysis followed the usual profile (7), the majority of the initial activity being

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,~

I' I'

7\\ I ' I ' I ' I '

\

"\

t I

'' \, ' '.....", \. , \A-\, ,, , 0

\

~

\ \ \ \ \ \ \

\

FIG. 2 Sedimentation velocity distribution of LPS-sensitive mature lymphocytes and precursor cells. Bone marrow fractions were stimulated with LPS and assayed for the initial number of lymphocytes by limiting-dilution analysis (~). ~e response of the lymphocytes produced in each fraction cultu~ed for 6 days at 1 x 10 cells/ml with LPS was measured in secondary cultures at 1 x 10 cells/ml (5 days for IgM-PFC's) ( . ) and at limitingdilution for 6 days (0). at 3 mm/h, a broad shoulder from 3.5 to 5.5 mm/h and a small peak at 6 mm/h. After the 5-day preculture period, the distribution of LPS-sensitive cells had changed, whether it was determined by Ig-PFC's or by limiting-dilution analysis. In both cases, some activity remained around 3.0 mm/h but a relatively sharp peak arose between 4.5 and 5.0 mm/h. As AE and LPS did not appear to stimulate entirely overlapping cell populations, AE stimulating younger cells, the number of new lymphocytes generated from precursor cells during a 7 day period of preculture with AE was measured in the following experiment. Normal bone marrow cells were fractionated and the response of LPS-sensitive lymphocytes initially present estimated by 6-day limiting-dilution cultures and from IgM-PFC's in mass cultures. The response of the cells capable of ultimately differentiating to IgM secretion under sequential mitogen stimulation (mature lymphocytes and precursor cells) was determined under similar conditions except that AE was present for 12 days and LPS only from the 7th day and the cultures read after 12 days. The number of lymphocytes found in each fraction after a period of 7 days of culture at 1 x 10 4 cells/ml with rat thymus cells and AE was measured directly in secondary limiting-dilution cultures with LPS. As seen in Figure 3, the frequency of responding cultures increased significantly in three regions of the gradient between 6 and 12 days and with the. addition of the AE. As in the case of LPS stimulation, the distribution of secondary culture lymphocytes differed from the initial distribution. It also appeared to differ from the distribution of the secondary culture lymphocytes detected with LPS stimulation. Moreover, the distribution measured depended partly on the assay used. The measurement of Ig-PFC's on day 12 yielded only one homogeneous peak at 4.0 mm/h while the cumulative limiting-dilution assay revealed the same peak at

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•

t FIG. 3 Sedimentation velocity distribution of AE- and LPS-sensitive mature lYlfphocytes and precursor cells. Bone marrow fractions were cultured with LPS at 1 x 10 cells/ml for 6 days to measure their IgM-PFC response ( . ) and at limiting dilution tQ estimate the initial number of lymphocytes ( They were also cultured at 1 x 10 cells/ml with AE for 7 days and either stimulated with LPS and assayed on day 12 for IgM-PFC's ( • ) or harvested and redistributed with LPS in secondary limiting-dilution cultures to be assayed 5 days later ( ~ ).

0).

4.0 mm/h but also a more heterogeneous one between 5 and 6 mm/h and possibly a third one at 7 mm/h. More direct and quantitative information regarding the origin of newly-formed LPSsensitive lymphocytes was obtained through the analysis of the progeny of single cells following the protocol described in Figure I, Bl and B2. Bone marrow cells were fractionated and the pools of cells cultured at limiting-dilution with LPS. The number of responding cells was measured after a continuous incubation of 6 days (mature lymphocytes) or 9 days (mature lymphocytes plus precursor cells). Individual cultures were recovered from other 10 ul microwells after 3 days, mixed with fresh rat thymus cells and LPS and redistributed amongst 60 microwells to measure by limiting dilution, the number of lymphocytes present in single original cultures. The results (Table 3) first indicate that the frequency of mature B lymphocytes was well below one per culture, except for the pool of cells sedimenting at 3.0 mm/h. The number of lymphocytes present in individual primary cultures on day 3 paralleled fairly well the frequency measured on day 9 rather than the one measured on day 6. Large numbers of mature cells were cultured from the 3 mm/h pool and at least some of the newly-formed cells must have arisen from cultures that initially contained mature cells. In other fractions however, specially in the 4 mm/h and the 7 mm/h pools, the frequency of cultures containing lymphocytes after 3 days of primary culture was such that some must have arisen from cultures that did not initially contain mature lymphocytes. The average

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TABLE 3 Clonal Differentiation of Precursor Cells during 3 days under LPS Stimulation Velocity (mm/h)

Initial number of respondini) cells/culture

Number of day 9 responding cells/culture a)

3

0.85

1.1

12, 10, 10, 5, 3 2, 2, 2, 0, 0

4

0.29

0.5

30,9,6, 3, 2 2, 1, 0, 0, 0

5

0.03

0.2

1, 1, 1, 0, 0 0, 0, 0, 0, 0

6

0.02

0.1

0, 0, 0, 0, 0 0, 0, 0, 0, 0

7

0,02

0.4

14, 2, 1, 1, 0 0, 0, 0, 0, 0

8

0,07

0.1

5, 3, 3, 0, 0 0, 0, 0, 0, 0

Number of lymphocytes/b~lture

on day 3

a) Determined by uninterrupted limiting-dilution cultures for 6 (initial) or 9 days with LPS. b) Limiting-dilution cultures were set up (20 cells/culture) and stimulated with LPS. After 3 days, individual cultures were harvested and redistributed into another 60 wells with LPS for limiting-dilution analysis. IgM-negative supernatants were scored after a further 6 days.

number of lymphocytes per individual responding culture was approximately 8 in the pools at 3 and 4 mm/h and around 4 in the two pools of largest cells. Since AE appears to stimulate large numbers of cells younger than those stimulated by LPS, a similar experiment with AE and LPS was performed with primary culture periods of 4 and 11 days at limiting dilution. As can be seen in Table 4, cultures were set up at a higher concentration and were above limiting-dilution for the first 2 pools as determined from day-4 Ig-PFC's or from the presence of IgM in the supernatant of the cultures on day 6. When secondary cultures were made on day 4, all the cultures from the 3 and 4 mm/h pools contained high numbers of lymphocytes whereas only a small fraction of the larger cells did. When recultured on day 11, all fractions still contained lymphocytes but their numbers had decreased by an important factor in the 3 and 4 mm/h pools while the frequency of positive cultures and the number of lymphocytes per culture had increased in the other fractions. The pools at 5 and 7 mm/h contained the highest numbers, an average of 6 lymphocytes per culture and all the cultures were positive. The majority of corresponding cultures had appeared negative when recultured on day 4.

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TABLE 4 Clonal Differentiation of Precursor Cells during 4 and 11 days under AE and LPS stimulation Velocity mm/hour

IgM-PFC/ cu Iture a)

Initial number b ) of mature lymphocytes per culture

Number of lymphocytes per culture after) 4 daysc

Number of lymphocytes per culture after 11 daysc)

3

200, 100, 87, 65 65, 15, 14, 11 10,8

5

30 d)

1, 1, 1, 1, 2, 3, 2, 4,3

4

140,85,46, 57,40, 34, 2, 2, 12, 10

5

30 d)

0, 0, 0, 1, 2, 2,4

5

12, 19, 4, 2, 6,5,1,3, 2, a

0,8

26, 0, 0, 0, 0, 0, 0, 0, 0, a

2,2,4, 5, 5, 6, 6, 8, 7, 14

6

2, 0, 1, 2, 7,1,0, 1, 8, 3

0.6

11,1,1, 0, 0, 0, 0, 0, a

0, 3, 1, 1, 2, 2, 2, 3, 5, 6

7

0, 2, 1, 4,2,0, 8, 0, 0, 1

0.6

2, 1, 1, 0, 0, 0, 0, 0, 0

°

3, 3, 2, 3, 2, 3,

6, 7, 8, 10

a) Cultures were set up at 50 cells/culture, stimulated with LPS and AE and individual 10 I cultures were assayed on day 4 for IgM-PFC's. b) Determined from IgM-negative culture supernatants on day 6. c) Cultures set up at 50 cells/culture with LPS and AE were harvested individually on day 4 or on day 11 and redistributed into 30 cultures. Culture supernatants were tested for IgM, 6 days later. d) All 10 cultures tested contained more than 30 lymphocytes or had too much IgM in the supernatant, the secondary cultures were all positive.

DISCUSSION AND CONa....USION The measurement of the number of mature, LPS-sensitive lymphocytes generated in vitro from precursor cells brings new information on the nature of these precursors and on their capacity for proliferation and differentiation. First of all, the results described above support previous indications that many different populations of precursors of B lymphocytes exist in normal bone marrow (7) and they were obtained using direct assays for lymphocytes. As found before, at least two LPS-dependent and three AE-dependent populations appear capable of generating new lymphocytes in vitro. The precursor cells differ in size and in their kinetics of differentiation to B lymphocytes. The estimates done by reculture of the number of lymphocytes produced

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from a given cell population indicate more precisely when the cells reach "maturity", what the clone size is, and show directly that LPS-sensitive cells arise from previously LPS-insensitive cells. Since the period of IgM-secretion of the cells of a clone is not synchronized and it is spread over many days, it is difficult to interpret in a simple manner the results of the secondary cultures done at early times of fractions already containing mature B lymphocytes. Indeed, even after a few divisions, mature cells may still have had enough proliferative capacity and may have given rise to enough IgMsecreting cells to yield positive limiting-dilution cultures. The early increase seen in the 3 mm/h pool in all experiments, for example, was probably due at least partially to this phenomenon. On the other hand, small lymphocytes may also have a significant capacity for self-renewal under conditions which encourage proliferation rather than maturation (15). This could also explain part of the increase in the number of "newly-produced" lymphocytes measured in the 3 mm/h pool which might rather be called "LPS-memory cells". The results of the reculture of pools of cells sedimenting faster than 4 mm/h appear simpler to interpret. It has already been found that the kinetics of the response of these fractions is slower than that of mature lymphocytes and that the frequency of early-responding cells is lower there than in the pools of small cells (7, 16, 17). Upon secondary culture, many more fractions contain mature lymphocytes than there were when initially cultured, which suggests that these lymphocytes have differentiated from immature cells. At least four different populations of precursor cells appear to be resolved under those conditions. The first two are detected in the presence of LPS and at relatively early times as the number of lymphocytes increased in the region of 4 and 7 mm/h. From the number of lymphocytes measured in single cultures, one might conclude that precursors at 4 mm/h began dividing soon after they were put in culture since they had time for an average of 3 divisions. The differentiation of precursor cells in fractions of large cells might have longer to go before reaching maturity, since fewer lymphocytes were found per culture and these could have arisen after only one to two divisions. The addition of the AE reveals the presence of 2, possibly 3 other populations of precursor cells. One sediments around 4 mm/h, produced LPS-sensitive cells at late times (up to 11 days) and appeared to differ in size from the population of LPS-sensitive cells detected in this region. The different sensitivity of the two populations to inactivation by anti-IgM and complement also suggests that they were not the same (18) and the AE-sensitive cells could be described as resting pre-B cells. The other population is more heterogeneous and is spread from 5 to 6 mm/h. Its distribution is similar to that of a population of cycling cells (19) and suicide experiments appeared to support this hypothesis (18). This precursor cell population had properties one would expect of cycling pre-B cells. The distribution of the third population peaked around 7 mm/h and it was not well defined because its response was low. It showed up only as one point in Figure 3 but significant numbers of LPS-sensitive lymphocytes were recovered from these cultures as seen in Table 4. As the detection of these cells appeared to depend on the presence of AE and they differentiated late to LPS-sensitive cells, they may represent another physiologic state of pre-B cells or possibly another stage of differentiation from stem cells. An early respor:lse in some experiments appeared to derive from cell aggregates. Altogether, since these experiments were done at limiting dilution which changed with time, background problems are not the same as in other systems (14), and these results show more directly and conclusively that mitogensensitive cells can be produced in vitro from immature precursor cells. It is obvious that the initial time of division and the number of divisions calculated above must be underestimated, since it was always assumed that all cells were recovered from the cultures and that they all survived the manipulations, which is unlikely to be the case. Another reason why the number of lymphocytes measured in single cultures cannot be taken as an accurate estimate of clone size is that measurements probably were not made at exactly the optimal times, i.e. when cells with a given functional property had stopped dividing. On the other hand, as the progeny of single cells can be analysed, this approach may provide a model system to investigate when differentiating precursor cells lose the potential for antibody diversification.

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REFERENCES 1.

YANG, W.C., MILLER, S.C., and OSMOND, D.G. lymphocytes. J.Exp. Med. 148: 1251, 1978.

2.

LAFLEUR, L., MILLER, R.G., and PHILLIPS, R.A. A quantitative assay for the progenitors of bone marrow-associated lymphocytes. J. Exp. Med. 135: 1363, 1972.

3.

LEVITT, D., and COOPER, M.D. Mouse pre-B cells synthesize and secrete u heavy chains but not light chains. Cell 19: 617, 1980.

4.

METCALF, E.S., SCHRADER, F., and KLINMAN, N.R. Murine models of tolerance induction in developing and mature B-cells. Immunol. Rev. 43: 143, 1979.

5.

ANDERSSON, J., COUTINHO, A., LERNHARDT, W., and MELCHERS, F. Clonal growth and maturation to Ig secretion in vitro of every growth-inducible B lymphocyte. Cell 10: 27,1977.

6.

GRONOWICZ, E., and COUTINHO, A. Selective triggering of B cell subpopulations by mitogens. Eur. J. Immunol.~: 771, 1974.

7.

DESLAURIERS-BOISVERT, N., MERCIER, G., and LAFLEUR, L. Size separation and polyclonal activation to immunoglobulin secretion of early precursors of B lymphocytes. J. Immunol. 125: 47, 1980.

8.

MILLER, R.G., and PHILLIPS, R.A. Separation of cells by velocity sedimentation. J. Cell. Physiol. 73: 191, 1969.

9.

KINCADE, P.W., RALPH, P., and MOORE, M.A.S. Growth of B lymphocyte clones in semisolid culture is mitogen dependent. J. Exp. Med. 143: 1265, 1976.

10.

ANDERSSON, J., COUTINHO, A., and MELCHERS, F. Frequencies of mitogenreactive B cells in the mouse. J. Exp. Med. 145: 1511, 1977.

11.

LEFKOVITS, I. Induction of antibody-forming cell clones in microcultures. Eur. J. Immunol • .f: 360, 1972.

12.

JERNE, N.K., and NORDIN, A.A. Plaque formation in agar by single antibodyproducing cells. Science 140: 405, 1963.

13.

MOLINARO, G.A., MARON, E., EBY, W.C., and DRAY, S. A general method for enumerating single cells secreting antigen: albumin-secreting hepatocytes detected as plaque-forming cells. Eur. J. Immunol.,2: 771, 1975.

14.

In vitro LAU, C.Y., MELCHERS, F., MILLER, R.G., and PHILLIPS, R.A. differentiation of B lymphocytes from pre-B cells. J.Immunol. 122: 1273, 1979.

15.

MELCHERS, F., COUTINHO, A., HEINRICH, G., and ANDERSSON, J. Continuous growth of mitogen-reactive B-Iymphocytes. Scand. J. Immunol.~: 853, 1975.

Maturation of bone marrow

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16.

PRESS, J.L., STROBER,S., and KLINMAN, N.R. Characterization of B cell populations by velocity sedimentation, surface Ia antigens and immune function. Eur. J. Immunol. 2: 329, 1977.

17.

HOWARD, M.C., and FIDLER, J.M. Antigen-initiated Blymphocyte differentiation. Austr. J. Exp. BioI. Med. 55: 501, 1977.

18.

DESLAURIERS-BOISVERT, N., MERCIER, G., and LAFLEUR, L. In preparation.

19.

PHILLIPS, R.A., and MILLER, R.G. Antibody-producing cells: analysis and purification by velocity sedimentation. Cell Tissue Kinet.l: 263, 1970.

Received for Publication September 1980 Accepted for Publication May 1981