Immunology, 1975, 28, 27 1.

The Immune Response in Stationary Suspension Cultures Containing Different Numbers of Cells THE SURFACE DENSITY EFFECT A. E. GURVITCH, AsIYA KORUKOVA AND OLGA GRIGORYEVA Laboratory of Chemistry and Biosynthesis of Antibodies, the Gamaleya Institute for Epidemiology and Microbiology, Academy of Medical Sciences, Moscow, U.S.S.R.

(Received 27 March 1974; accepted for publication 23rd May 1974) Summary. Correlation between the number of dissociated spleen cells, incubated with antigen in a modified Mishell and Dutton system, and the number of antibodyforming cells (AFC) produced as a result of incubation has been studied in mice of C57B1/6 strain. It has been shown that when suspension densities are increased 2- to 4-fold, the number of AFC formed is very often reduced 10- to 100-fold, although the percentage of viable cells recovered at the end of incubation is not significantly diminished. The observed reduction of AFC formation in cultures containing increased numbers of cells, designated here the surface density effect, was found to be expressed more by spleen cells of unimmunized than of immunized mice. Inhibition was dependent on the thickness of the cell layer formed on the bottom of the incubation vessel (cells per square centimeter), rather than on the cell: volume ratio of cultures. The effect was not due to a deficit of antigen or nutrition and could not be reproduced by adding of incubation media conditioned by dense cultures. It was determined not by impairment of clone induction but by inhibition of subsequent proliferation. This suppression is reversible and can be reversed by decreasing the cell density, even after 3 days of culture. INTRODUCTION Analysis of the antibody production process has been greatly facilitated by development of methods permitting induction of a primary immune response in vitro (Mishell and Dutton, 1967; Click, Benck and Alter, 1972; Claman and Mosier, 1972). A paradoxical discrepancy has been revealed by previous studies in this and other laboratories. If the intact spleen is isolated from the organism during the period of declining secondary immune response and perfused in vitro, the decline in antibody formation continues in much the same manner as before isolation (Gurvitch and Nikolaeva, 1971a, b; Atkins, Robinson, Trimble and Eisman, 1970). If a secondary response is induced in the isolated perfused spleen, it is extremely poor (Atkins et al., 1970). In contrast, if non-fractionated spleen cell suspensions are prepared from immune spleens after either primary or secondary immunization, and incubated in vitro, the decline in the antibody formation is delayed for some time. On adding antigen to such suspensions, Correspondence: Dr A. E. Gurvitch, Laboratory of Chemistry and Biosynthesis of Antibodies, Gamaleya Institute

for

Epidemiology and Microbiology, Academy of Medical Sciences, Moscow, U.S.S.R.

271

A. E. Gurvitch, Asiya Korukova and Olga Grigoryeva 272 the intensity of antibody biosynthesis is greatly increased, becoming many times higher than in the intact organism (Dutton and Mishell, 1967; Pazdernik and Uyeki, 1973; Gurvitch, Grigoryeva and Korukova, 1974; Gurvitch and Sidorova, 1964). These differences in the course of the immune process did not seem to be related to proportions of cells participating in the systems compared (B and T lymphocytes, A cells), since intact spleen and non-fractionated cell suspensions prepared from it are not much different in this respect. The present study has been devoted to defining the influence of the number of cells interacting in cultures upon the development of the immune process. Preliminary data have been published already (Korukova, Grigoryeva and Gurvitch, 1964).

MATERIALS AND METHODS Experiments were made with cell suspensions prepared from spleens of C57B1/6 mice. The antibody-producing system was that of Mishell and Dutton (1967) as modified by Click et al. (1972) and to a small extent by us (Gurvitch et al., 1974). Sheep red blood cells (SRBC) or a water-soluble antigen (WSA) were prepared from them according to the method of Seman, Marie and Bussard (1971) and were used as the antigen. Spleens were removed aseptically from mice, minced and disintegrated in cooled Eagle's medium. Cell clumps and debris were allowed to settle for 10 minutes, and the supernatant cells were washed twice with Eagle's medium using centrifugation for 10 minutes at 700 g. Final suspensions were prepared in the medium recommended by Click et al. (1972) which we supplemented with insulin (0'24 u/ml) and glucose (1.0 mg/ml). Suspensions containing 5 x 106, 10 x 106, and 20 x 106 spleen cells per millilitre were dispensed in 1-ml quantities into culture vessels which were then filled with gas mixture (5 per cent CO2: 10 per cent 2: 85 per cent N2), sealed with rubber stoppers, and incubated for 1-5 days at 370. In some experiments the culture volume was varied from 0 5 to 2-0 ml. The culture vessels were 15-ml flat-bottomed flasks of 20 mm diameter. The flasks had been siliconized with 'Antifomsilanum' (Chemico-Pharmaceutical Plant No. 3, Riga). Following incubation, each culture was examined for the number of surviving cells using staining with eosin-trypan blue mixture. The antibody-forming cells (AFC) were assayed by two methods: the direct method of Jerne and Nordin (1963) for IgMproducing AFC, and the indirect method of Sterzl and Riha (1965) for IgG-producing AFC. Each determination was made on four parallel cultures, and the results are expressed as the mean numbers of AFC/106 recovered viable cells. To compare nucleoside and amino acid uptake in cultures containing different numbers of cells, [3H]thymidine was added to cultures at the beginning of incubation, and ['4C]glycine was added 8 hours before termination of cultures. Cells were washed with Eagle's medium and then with 5 per cent trichloroacetic acid, and radioactivity was measured using the Intertechnics Type SL-40 liquid scintillation counter.

RESULTS A well expressed primary immune response to SRBC and WSA could be obtained in our experiments only by using siliconized flasks and mercaptoethanol-containing medium. If either of the two factors was absent the response did not develop.

Immune Response in Stationary Cultures 273 The number of AFC attained at the end of incubation was dependent on the number of spleen cells taken for incubation. A certain initial number of cells was optimal to ensure intensive AFC formation, and if this was increased 2- or 4-fold, the AFC resulting were greatly reduced. The reduction was apparent in all cases, although variable: there were experiments in which the AFC numbers were reduced 4- to 10-fold, but in the majority of cases the decrease was greater, 50- to 100-fold (Table 1). TABLE I

CORRELATION

BETWEEN THE INITIAL NUMBER OF SPLEEN CELLS IN THE CULTURE AND THE NUMBER OF ANTIBODY-FORMING CELLS (AFC) PRODUCED

Experiment number 3-19 3-29

3-30

3-37

Antigen

SRBC SRBC SRBC SRBC SRBC SRBC WSA WSA WSA WSA WSA WSA

Initial numbers of cells/culture (x 106) 5 10 20 5 10

20 5

10 20 7-5 15 30

Immune response on the 4th day

AFC/ 106 surviving cells

AFC/culture

672

880

439 9 1314

1360 42 1085

1660 481 818 197 7 469 97 5

3420 1142 1128 570 65 1090 353 40

Cell viability (per cent) 28 29 18 17

21 13 29 30 45 31 24 27

It is of interest to note that, as a rule, the reduction was greater and was apparent with smaller initial cell numbers if WSA was used as the antigen, rather than SRBC. Since the method ofJerne and Nordin we used revealed only IgM-producing cells, the observed reductions could be due to some shift in AFC from IgM to IgG synthesis. However, determinations of IgG-producing cells by the indirect method showed no increase in such cells in cultures with increased initial cell numbers. The reduced immune response in cultures with increased number of spleen cells was not accompanied by significant impairment of cellular viability. In experiments employing different initial cell numbers the percentage of cells recovered at the end of incubation was nearly the same in all groups. In other experiments, cultures with larger numbers of cells exhibited somewhat lower viability, but the relative reduction in total viable cells was very much less than the inhibition of AFC formation. The kinetics of the immune responses and cell viability in cultures containing different numbers of cells are shown in Fig. 1. It is evident that the response followed an exponential curve when the initial cell number was optimal (5 x 106 cells), and that cultures prepared with 10 x 106 and 20 x 106 cells had considerably lower rates of increase of AFC numbers which did not increase with time. The relative inhibition of responses became, therefore, progressively greater. In the experiment shown the relative inhibitions on days 2, 3, and 4 in cultures with 20 x 106 cells were 71, 80, and 94 per cent, respectively. An obvious question arose whether the observed phenomenon was associated with increased cellular concentration (cells per millilitre of culture medium) or with increased surface density, i.e. the thickness of the cell layer formed in culture flasks (cells per square

A. E. Gurvitch, Asiya Korukova and Olga Grigoryeva 274 centimeter of the bottom). To clarify this point, experiments were made in which both the number of cells per flask (5, 10 and 20 million) and the volume of the medium in which they were suspended (1.0 and 2-0 ml) were varied. The results (Table 2) showed a significant effect of surface density; in samples which did not differ in concentration of spleen cells, the AFC numbers diminished abruptly as the surface density was increased. Ia>

= I

L-I to

-

(): a) I=_ (At U

0

0

0

E 0 IL

Days of culture

FIG. 1. Kinetics of (a) cell viability and (b) AFC formation in the course of incubation of samples containing different numbers of spleen cells. (o) 5 x 106 initial cells per flask. (-) 10 x 106 initial cells per flask. (A) 20 x 106 initial cells per flask. TABLE 2 EFFECT OF THE CONCENTRATION AND SURFACE DENSITY OF CULTURED CELLS UPON THE NUMBER OF AFC

Group number 1

2 3

Initial numbers

cells/culture (x 106) 5 5 10 10 20

20

Volume of medium (ml) 2-0 1.0 2-0

1*0

2-0 10

Surface

density (cells/cm2) (x 106) 159 1-59 3-18 3-18 6-36 6-36

Concentration

AFC/ 106 cells

(cells/ml) (x 106) 2-5 50 50 10 0 10 0 20-0

713 818 161 197 13 7-3

A series of experiments was made using spleen cells from unimmunized and immunized mice (Table 3). These showed that suppression of the immune response by greater initial cell numbers was less evident in cultures prepared from spleens of immunized animals. In the case of mice immunized 3 days before removal of spleens, the inhibition of AFC formation was 24 times less than with spleen cells from unimmunized animals. It is of

Immune Response in Stationary Cultures

275

TABLE 3

SUPPRESSION OF AFC FORMATION IN CELL SUSPENSIONS OF DIFFERENT DENSITY PREPARED FROM SPLEENS OF UNIMMUNIZED AND IMMUNIZED* ANIMALS

'Direct' AFC

Initial

Spleen donors Unimmunized mice Mice immunized 1-day previously Mice immunized 3 days previously Mice immunized 6 days previously Mice 10 days previously

cells/

culture (x 106)

AFC/ 106 cells

Percentage

5 10 20

515

100 6-6 0-68 100 27-2 2-2 100 43-5 16-5 100 38-8 5*9 100 35 0 8-9

5 10 20 5 10 20 5 10 20 5 10 20

34 3-5 2493 675 54 3821 1663 629 1716 660 101 1292 454 115

'Indirect' AFCt

AFC/ 106 cells

Percentage

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

2945 1051 387

100 35-6 13-0

* Mice were immunized intravenously with 500 x 106 SRBC. Cultures were stimulated with WSA

(50 p~g/ml).

t n.d. = Not determined.

interest to note that, in spite of the relative reduction of the response in 'dense' cultures prepared from the spleens of mice immunized 3 days before the experiment, the absolute numbers of AFC were, nevertheless, greater than those attained under optimal conditions with cells of unimmunized animals. When mice immunized 10 days previously were used, not only IgM- but IgG-producing AFC were formed in the suspensions. Formation of both AFC types was about equally inhibited by increased density of culture, but was again considerably less pronounced than in the spleen cell suspensions from unimmunized animals. It had to be ascertained whether the observed effect was due to insufficient antigen or nutrition in samples with increased numbers of cells. Experiments with dense cultures showed that even a 20-fold increase in antigen concentration had no effect on the degree of inhibition of AFC formation (Table 4, Group 1). To test the possibility of nutritional deficiency, an enriched medium was prepared. By comparison with the usual medium, it contained 3-fold quantities of the following components: essential and non-essential amino acids, vitamins, nucleic acid precursors, glutamine, sodium pyruvate, glucose and insulin. The enriched medium supported somewhat more AFC formation than the usual medium, but it was again observed that 20 x 106 initial cells formed almost 40 times less AFC than cultures with 5 x 106 cells (Table 4, Group 2). That the suppression of AFC formation was not connected with nutritional deficiency is shown by experiments with daily replacement of half the incubation medium by fresh medium. Such renewal at most only partly relieved suppression (Table 4, Group 5). Neither AFC formation nor its inhibition were significantly influenced by increased levels of foetal calf serum or mercaptoethanol, although both are important ingredients of the medium (Groups 3 and 4). E

276

A. E. Gurvitch, Asfya Korukova and Olga Grigoryeva TABLE 4

INDEPENDENCE OF THE SUPPRESSION OF AFC FORMATION IN DENSE CULTURES FROM VARIATIONS IN THE LEVELS OF ANTIGEN, INGREDIENTS OF EAGLE'S MEDIUM, MERCAPTOETHANOL, FOETAL CALF SERUM, AND FROM PARTIAL RENEWAL OF THE INCUBATION MEDIUM

Group

Experiment

number

number

Factor studied

AFC formation in the cultures

Maximum

with initial cell numbers of:

suppression

5X106 1

2

3 4

5

3-35 3-37

3-36* 3-37

3-33*

Antigen concentration 296 5jug/ml WSA 50 ,pg/ml WSA 677 100 ug/ml WSA 584 Ingredients of Eagle's medium (see text) Usual concentration levels 469 3-fold concentration levels 840 Mercaptoethanol concentration -5M 1*7 x 10 180 50 X10-5 M 163 150 x10-5 M 273 Foetal calf serum concentration 10°/ 469 20°/ 304 383 30%o Partial renewal of incubation medium Without renewal 301 1/2 medium renewed daily 298 *

Group

Cultures

rnber

were

ntial cl Is

flask (x 0) per

20X106

(percent)

152 366 296

17 71 35

94 89 94

97 391

5

23

99 97

23 33 36

87 80 87

5

97 97 44

9 5

99 97 99

125 198

76 88

75 70

incubated for 3 days only.

AFC formation per 106 cells

Operations

L1

5

10 X 106

2

20

U

3

20

U

4

20

0 L S)

5

5

Li

6

5

g

7

20

L)

;1

30 0

76

306

1 1

298 88

2 I

0

1

2

3

Days of culture FIG. 2. Failure to transfer the suppressive effect with culture media conditioned by dense suspensions or to alleviate it by replacements with fresh medium. The arrows indicate replacement of half the medium with fresh medium.

Immune Response in Stationary Cultures 277 Several experiments were made in an attempt to transfer the suppressing effect with conditioned incubation media. Results of one such experiment are presented in Fig. 2. Using the usual procedure, flasks containing 5 x 106 cells produced 4 times the number of AFC produced in flasks containing 20 x 106 cells (Groups 1 and 2). (The relatively low inhibition was due to testing for AFC on the 3rd rather than the 4th day of culture, which, as already mentioned, showed less marked differences between cultures of different densities). In a group of flasks containing 5 x 10 cells (Group 5), half of the medium was changed after 24 or 48 hours of cultivation for medium in which 20 x 106 cells had grown for the same time (Groups 3 and 4). This produced no inhibitory effect. Neither was suppression observed after similar replacements of the incubation medium with medium conditioned by growth of 10 x 106 cells. nihtal cells

Aterations

per flask

in density

AFC

formation per 06 cells

xK)6) 20

-481

--

390

l0

1760 0

2

3

4

Days of culture

FIG. 3. Effect on AFC formation of changing the surface densities of cultures after the 1st day of incubation.

The experiment described included several control determinations. To exclude possible effects of the substitution procedure itself, half of the medium in some flasks was twice replaced by fresh medium (Groups 6 and 7). The replacement was invariably accompanied by shaking of samples, cooling during treatments outside the incubator, centrifugation (700 g), and repeated saturation with the gas mixture. It was therefore ascertained that these manipulations did not result in detectable reduction of AFC formation. To exclude distorting effects, control experiments were carried out in which cultures were manipulated, but without actual substitution of medium. An important question was whether the observed phenomenon involved the induction phase of antibody formation or whether it mainly reflected retarded proliferation of induced AFC clones. To decide this, experiments were done in which the surface density was changed 24 hours after the start of incubation, in accordance with the contention (Dutton and Mishell, 1967) that the induction phase is essentially over within the 1st day of incubation and that proliferation of the established cell clone occurs during subsequent days. In one such experiment, shown in Fig. 3, SRBC were used as the antigen. Cultures

A. E. Gurvitch, Asiya Korukova and Olga Grigoryeva 278 containing 20 x 106 cells attained an average of 481 AFC by the end of incubation, while those with 10 x 106 cells produced 1660 AFC. Samples of cultures with 10 x 106 cells were combined by pairs after incubation with antigen for 24 hours; the culture volume was adjusted to an initial 1 ml, and incubation was continued for a further 4 days. The number of AFC produced in such double samples was approximately the same as in cultures containing 20 x 106 throughout the experiment. In another part of this experiment, samples containing 20 x 106 cells were divided in two after 24-hour incubation. The culture volume was adjusted with medium conditioned by lOx 106 cells, so as to avoid introduction of fresh medium. It was found that these cultures, which have had 20 x 106 cells during the first 24 hours and lO x 106 cells afterwards, produced about the same numbers of AFC as cultures which contained 10 x 106 cells from the beginning. _600

10,000

1500

0~~~~~~ t2

E

~8000 -

\\

400Q

1000 5

0

6000C

~~~~~~~300

p

~~~4000

C

E

500C I

2

L 3

200) 0

-2000

100 5

10

20

Nuimber of spleen cells per culture (x106) ) and [14C]glycine (- - -) incorporation with AFC FIG. 4. Comparison of [3H]thymidine ( formation (- - ) in cultures containing different numbers of spleen cells.

The conclusion that increased surface density results in suppressed cellular proliferation was also supported by observations on [3H]thymidine incorporation. It was found in preliminary experiments that [3H]thymidine added to cultures at the beginning of incubation (0 1 yuCi/ml final concentration) had no influence on AFC formation. When this quantity of the labelled compound was introduced into cultures of increasing density, the decline in AFC was closely paralleled by the curve of [3H]thymidine uptake (Fig. 4). Amino acid incorporation was determined during the last 8 hours of cultivation by adding [14C]glycine (1 0 uCi/ml final concentration). It can be seen that amino acid incorporation declined somewhat more slowly that the AFC numbers formed in cultures

of increasing surface density. It was of interest to find out what would be the effect on AFC numbers of altering surface density during the period of most active exponential growth between the 3rd and 5th days ofin vitro cultivation. In the experiment presented in Fig. 5, cultures with low density (3.9 x 106 initial cells) produced significant numbers of AFC by the 3rd day (736) which increased between the 3rd and 4th days (up to 1362) and then declined (Curve 1). Forty low density cultures were pooled on the 3rd day of incubation (Pool 1). Part of

Immune Response in Stationary Cultures 279 Pool 1 was used for preparing samples with quadruple number of cells. Another part of Pool 1 was used to prepare samples corresponding to the initial culture. The kinetics of AFC in the samples with unchanged density was much the same as in cultures containing the low cell number from the start of the experiment (Curve 2). A quite different picture was observed, however, in cultures prepared from the same cellular pool in which the density had been increased 4-fold; this prevented any further rise in AFC number

(Curve 3).

The opposite effect was obtained when dense cultures (Pool 2), containing 15 f6 x 106 initial cells were adjusted to lower cell numbers on the 3rd day of incubation. Following 4-fold dilution, these cultures showed increased AFC formation, becoming especially pronounced during the 5th day of incubation (Curve 6). The rates of AFC formation in dense controls, both intact and adjusted, remained slow and comparable (Curves 4 and 5). The results of the latter experiment are additional evidence that the incubation medium of dense cultures contained by the 3rd day no extracellular factors which could be responsible for slow AFC formation.

a,8

=

Pool 1 (low density cultures)

I000~~~~~~ 0

500_ Pool 2 (high density cultures)

3

4

5

Days of culture FIG. 5. Effect of surface density, altered on the 3rd day of incubation, on AFC formation. (e) Curve 1. AFC in control samples with 3 9 x 106 initial cells. (a) Curve 2. AFC in samples prepared on day 3 from the common pool 1 (density corresponding to 3-9 x 106 initial cells). (A) Curve 3. AFC in samples with increased (4-fold) density prepared on day 3 from the common pool 1 and corresponding to 15-6 x 106 initial cells. (A) Curve 4. AFC in control samples with 15-6 x 106 initial cells. (El) Curve 5. AFC in samples (corresponding to 15-6 x 106 initial cells) prepared on day 3 from the common pool 2. (-) Curve 6. AFC in samples with decreased density (corresponding to 3 9 x 106 initial cells) prepared on day 3 by dividing one dense culture into 4.

DISCUSSION The reported experiments characterize some aspects of an inhibitory effect dependent upon the number of participating spleen cells and capable of diminishing the magnitude of an in vitro immune response. An increase in cultured cells beyond optimum brings about a rapid decline in the number of AFC formed during incubation, although the viability of cells remains unchanged or is only slightly impaired. Very often, a 4-fold

Korukova and Olga Grigoryeva A. E. Gurvitch, increase in incubated spleen cell numbers resulted in10- to 100-fold reduction in AFC. The suppression of AFC formation caused by increase in numbers of cultured cells was greatest with cell suspensions from unimmunized animals. With spleen cells from immunized mice, the effect was considerably less, especially if the immunizing injection 280

Asiya

had been given 3 days previously.

Observations on AFC formation in cultures differing in both number of cells settling down per unit of the flask bottom area and their concentration indicated that suppression was more dependent on surface density (Table 2). If the average diameter of spleen cells is assumed to be about 16 Pm, the number of cell layers formed on the bottom of the culture flask can be estimated. Such calculation gives three layers of cells for cultures containing 5 x 106 initial cells, and six and twelve layers for10 x106 and 20 x106 cells, respectively. The less intensive AFC formation in cultures containing more numerous cells could not be explained by a deficiency in antigen, since even 20-fold variations in antigen level failed to influence the effect. The reduction was little dependent on the volume of the medium (Table 2) and was not connected with the supply of amino acids or other nutrients essential for cellular vital activities (Table 4). Neither can it be explained by impaired access of oxygen or other nutrients to the lower cell layers, since the effect seemed to involve not only the lower layers but the upper ones as well. This is shown by the degree cells (Table 1). of total AFC reduction per flask in cultures containing 5 x 1 and 20 x The reduction of AFC formation was not connected with impairment of the induction is supported by the fact that a decline phase of the immune response. This ifconclusion in AFC numbers could be produced the surface density of cultured cells was increased after 1 and 3 days of incubation, that is, at a time when the induction phase is already over (Dutton and Mishell, 1967). A number of findings point to inhibition of proliferation in samples containing increased incorporation in samples with numbers of cells: (1) the abrupt decline in in growth of AFC numbers arrest elevated surface density (Fig. 2); (2) the complete which had previously in cultures increase to density following adjustment of cell numbers in AFC in increment much lower the daily in a rise exhibited rapid AFC (Fig. 5); (3) dense cultures compared with cultures with fewer cells, although the induction phase has already taken place in both (Figs 3 and 5); (4) the reversible character of the effect. A high degree of reversibility is demonstrated by the fact that a 4-fold dilution of density-inhibited cultures on the 3rd day of cultivation resulted in greatly accelerated formation of AFC, due to their intensive proliferation (Fig. 5). Retarded proliferation of AFC in dense cultures cannot be attributed to accumulation in the medium of any 'long-lived' inhibitory substance. This is indicated by: (1) failure to transfer the effect with medium conditioned by dense suspensions; (2) failure to abolish or significantly reduce the effect when increased culture volumes or daily renewals of half of the medium were used. Neither can the effect be connected with accumulation of antibodies in the cellular environment, since AFC numbers and, hence, antibody concentrations in dense cultures were, as a rule, considerably lower than those in cultures with fewer cells. It is not possible at present to propose any definite mechanism for this 'surface density effect'. It may be connected with local accumulation of a rapidly degraded inhibitory surfaces of participating cells, as postulated for cell metabolite, or with direct contact of (1973). position determinants by McMahon

O6

[3H]thymidine

106

281 Immune Response in Stationary Cultures The 'surface density effect' may be one of the factors determining the marked differences in IgM synthesis in vitro which are observed between perfused intact spleen, on the one hand, and suspensions of dissociated spleen cells, on the other. The effect seems to be very similar to contact inhibition (Abercrombie and Heysman, 1954; Stoker, 1972; Abercrombie, 1970; Vasiliev and Malenkov, 1968). Under the conditions of our experiments, however, with siliconized flasks and suspensions of spleen cells, there was no attachment of cells to the bottom of the flask and no monolayer was formed. The reported observations seem to underline one aspect of the cellular interactions involved in control of the immune response. This is that the interactions depend not only on the proportions between different cell types (B and T lymphocytes, A cells) but also on the number of cells taking part in the induction of antibody formation.

ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr A. Y. Friedenstein, L. N. Mechedov and Dr E. A. Luria for useful suggestions. REFERENCES

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