Immune responses in vitro. I. Culture conditions for antibody synthesis

CELLULAR

3, 264-276

IMMUNOLOGY

Immune

Responses In Vitro. I. Culture Conditions Antibody Synthesis 1

E.

ROBERT

(1972)

CLICK,

Medical

LORETTA

MicroDioloy~,

BENCK,

Uwixrsity Received

AND

BARBARA

of W’isconsis, Madison,

For

J.

ALTER

Wiscomix

June 23, 1971

The addition of mercaptoethanol to the culture medium and the optimization of other components has substantially improved the response of mouse spleen cells to in vitro immunization with heterologous RBC. As a result of these changes in the medium, the necessity of rocking and feeding cultures has been eliminated. The in vitro response of C,,Bl spleen cells was as much as 30 times greater than that achieved iu Go. This suggests the loss of an zlz viva control mechanism. Because the magnitude of responses was strain dependent, this culture system may facilitate the study of the genetic control of antibody synthesis.

INTRODUCTION

Of the immune events that an antigen can induce in an animal, three (humoral, cell-mediated, and tolerance) have been achieved with mouse cell suspensions in culture (l-9). Antibody synthesis has been attained in three types of culture systems-that described by Mishell and Dutton ( 1) and those described by Marbrook (2, 3). These tissue culture techniques provide a vital tool for the analysis of factors regulating cellular events in the immune response. This paper describes a fourth culture system which has several technical advantages over the systems introduced by Mishell and Dutton and by Marbrook. Parameters are presented which permit successful immunization of mouse cells cultured in an air-CO? atmosphere under stationary conditions without daily feeding. In addition, preliminary results suggest that an additional aspect of the immune response, the genetic control of antibody synthesis, can be analyzed in this culture system. MATERIALS AND METHODS

Mice. Two- to five-month old male inbred C,,B1/6J, Balb/C, and CBA mice from our own colony were used for all experiments. For any in vitro experiment the spleens from a minimum of three mice of the same age were pooled. Antigens. Sheep RBC (ARS Gibco, Madison, Wis.) of both high(H)-type (#105) and low(L)-type (1) were obtained in 50% Alsevers. Sheep RBC were washed twice in PBSCaMg [phosphate buffered saline (10) containing 0.18 mM CaCl, and 0.065 mM MgS04], once in a modified Eagle-Hanks medium (EHAA, r This work is supported in part by grants 20659) and from the Damon Runyon Memorial 264 0

197’2 by

Academic

Press,

Inc.

from the National Science Foundation (GBFund for Cancer Research (DRG-1043).

ANTIBODY

SYIGTFIESIS

IIS

VITRO

265

see test). pH 7.65, suspended at 2 X 109/ m l in modified EHAA, and stored at 4°C. Burro and horse RBC (BRBC and HRBC) were also obtained from ARS Gibco and prepared in the same manner as SRBC. BRBC were stored at 1 X lO!‘/ml and HKBC at 2 X lO”/ml. Cztlturr Mcdiztnt Reagents. The following reagents were purchased from Microbiological Associates (Bethesda, Md.) : MEM essential amino acids (50X ‘), 200 111~ L-glutamine (100 x ) _ ?VIEM vitamin mixture (100X ), and 100 lllM sodium and MEM nonessential amino acids pyruvate (100X ). Hanks l{SS (10X) (100X ) were either purchased from Microbiological Assoc. or prepared and sterilized 1,~ Millipore filtration. Other components of the culture medium were prepared as follows: nucleic acid precursors-l g/liter each of adenosine, guanosine, cytosine, and uridine ( 100 X ) ; lo* units/ml penicillin G and 10 mg/ml streptomycin sulfate (100X ) ; 4.4% (W/V) sodium bicarbonate; and 0.1 nf (0.7 ml/lOOml) mercaptoethanol (Mann Research Laboratory, New York, NY). The nucleic acid precursor and sodium bicarbonate stocks were sterilized by autoclaving the dry components prior to addition of sterile water and stored at 4°C. The antibiotic stock was sterilized by Millipore filtration and stored at - 20°C. Fetal calf serum was obtained from the Colorado Serum Company in Denver. .A11water used for reagent preparations was double distilled. Preparation of cells for czflfzrre. Mice were killed by cervical dislocation. Their spleens were minced aseptically with mouse tooth forceps in glass Petri dishes containing .3-S ml cold phosphate buffered saline (PBS) and transferred to 50 ml centrifuge tubes containing 3030 ml PBS (3-4 spleens/tube). Undissociated cells were allowed to settle 5-10 min at 4°C. The cell suspension (largely single cells) was then transferred to another tube and centrifuged at 3409 for 8-10 min. The cell pellet was suspended in PBS and recentrifuged under the above conditions. -Except ;\s noted in the text, the mouse RBC were removed by the following lysing procedure: the cell pellet was suspended in 0.4-0.6 ml PBS, to which 10-20 ml of glass distilled water was added with a pipette followed within 5 set by a sufficient amount of 10X PUS to return the solution to isotonicity. After this treatment, the cells were washed twice in PBS by centrifugation and resuspension ; the final volume was 0.5 ml PBS per spleen equivalent. Cell zdability. Cell counts were made on all cell suspensions prior to use and on recovered cells from all dishes after culture. Viability was assessed by the eosin exelusion technique ( 11) in which cells were diluted in a solution of PBS containing 3% bovine serum and 0.1% eosin. Cells which exclude eosin were counted in a hemocyt.ometer. C2llture conditions. Quadruplicate cultures of 7-g X 106 nucleated cells were incubated in 35 mm Petri dishes (Falcon Plastics, No. 3001) containing 2 ml medium. E.xcept as noted, 2 X 10’ H-type SRBC/dish (optimal concentration) were used as the antigen in all experiments. The stationary cultures were incubated at 37°C in a humidified atmosphere of 10% 0,-S% CO&S% NZ, (10% 0,) or 5% CO, in iair (air). HaellLolytic plaque assay. The cells from individual dishes were harvested by

266

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BENCK,

AND

ALTER

scraping the dishes with a rubber policeman, centrifuged, and suspended in 2 ml PBSCaMg. The number of plaque forming cells (PFC) per culture was determined by the Mishell and Dutton ( 1) modification of the assay originally described by Jerne. Nordin and Henry (12). Briefly, 10-100 ~1 of cell suspension and 2 drops of SKBC (2 X lO”/ml) were added to tubes containing 0.5 ml of 0.5% agarose at 4446°C. The mixture was poured onto microscope slides precoated with agarose and allowed to gel for 5-10 min. The slides were then incubated at 37°C in a humidified air incubator for 1 hr, followed by two additional hours of incubation in 10% fresh guinea pig complement (stored at -76°C). Duplicate assays were done for each culture. The plaques were counted with the aid of a dissecting microscope. All data are expressed as the mean number of PFC/lOa recovered cells that were viable. The standard error within each set of quadruplicate cultures was less than 15%. RESULTS

Variation of culture ingredients. Figure 1 shows the effect of various concentrations of mercaptoethanol (MET) in the medium on the number of PFC and on cell viability after 4 days of culture in both 10% 0, and air environments. At all MET concentrations, more PFC (ca. 50%) were induced in 10% 0, than in air, even though the cell viability was higher in air. The maximum number of PFC

FIG. 1. PFC formntio~z alzd cell viability nt van’ous cowelttsations of mrrmptoethaxol. C,,BI spleen cells plus SRBC were cultured 4 days in the medium described in Table 3. Mercaptoethanol was varied as shown. PFC obtained in air O--O and in 10% 0: A--A. Cell swviva1 in air C----O andin 10% 0, A-----A.

ANTIBODY

SYNTHESIS

IN

267

VITRO

was elicited at 5 X lo-” M in both gas environments. Higher concentrations depressed the response with little concomitant effect on viability except at 5 X 1(14 M, a conctentration at which the viability was repeatedly lower. Omission of ?v$ET severely reduced cell survival, and virtually no PFC were found. Comparison of the curves representin g PFC and cell survival in Fig. 1 suggests that the enhancement of PFC by MET is not merely a function of cell viability. Cell survival remained constant within the range of 1 X 10-j to 2.5 X lo4 M, which included a low response of 536 PFC/l@ at 1 X 10e5 n1 as well as the peak response of 3330 PFC/106 at 5 X lo-” M. The active moiety of MET was ascertained by testing its ethanolic derivative as well as unrelated sulfhydryl agents for their ability to enhance the response. As &own in Table 1, ethanol failed at both 10” and 2.5 X 10m5M, whereas all the reducing agents tested enhanced the production of PFC to some extent. The most effective agents were MET and dithiothreitol (DTT). Reduced glutathione resulted in PFC enhancement but, in general, not as effectively as DTT and MET. L-Cysteine showed a slight enhancement in only one experiment. While both MET and DTT resulted in comparable responses, MET n‘as selected as .the reducing agent for all further experiments because the molar concentration required for maxi:num activity was 10-fold lower than that of DTT (Table 1) and because DTT had a narrower range of optimal concentration in 10% 0, (1650 PFC/106 at 2.5 X lo4 M ; 5870 PFC/lO@ at 5 X 104 M ; and 2960 PFC/lO6 at 10.“M) than MET (Fig. 1). Figure 2 shows the number of PFC obtained in two different experiments at various concentrations of nucleic acid precursors in the medium. Although signifycant responses occurred in the absence of nucleic acid precursors, the maximum response in both experiments was attained with 25 mg/l. Higher concentrations inhib ited the response and depressed cell survival. TABLE IN VITRO

--

Compound

Mercaptoethanol Dithiothreitol Reduced glutathione L-cysteine Ethanol None

PFC FoI(~~ATIox Maximum effective concentration (Ml 5 x 10-j 5 x 10-d 2.5 x 10-Z 1x10-3c 1 x lo-” 2.5 x 10-s

1

m THE PRESI:NCX

OF MET

AND

RELATED

COMPOUNDS

-

PFC/lOG cells a 4-2 h

3-25

3-18 ~__10% 0~ air

3-19

10% 02

air

10% 02

air

10% OS

air

2950 3150 1340

2410 2620 -

3800 24.50 -

3530 ~

4020 3970 3930

3160 3660 ~

-

-

38

-

-

-

3925 5870 1230 200

3285 ~ -

l-------l------48

2

3.5

2

315

1

44

” Determined after four days of culturing CsTBL spleen cells plus SRBC in medium in Table 3. Reducing agents were varied as showu. 6 Experiment numbers. c This was the highest concentration teated due to its insolubility above 10-3~.

27 described

268

CLICK,

BENCK,

AND

ALTER

2-c I

-

2o

-0

I

0

I 20

I

I 40 MGOFNAPI

I

I 60

I

I 80

2

1 loo

L

FIG. 2. PFC formation and cell s~iability at various concclztratiow of nucleic acid precursors (N.4P). C,,Bl spleen cells plus SRBC were cultured 4 days in the medium described in Table 3. The concentration of nucleic acid precursors was varied as shown. PFC response (O- - -0) and cell survival (O---O) for experiment number 3-4. PFC response (U---a) for experiment number 12-31.

Table 2 shows the importance of nonessential amino acids (NEAA), L-glutamine, and calcium ions in the culture medium for PFC induction and cell survival. Spleen cells cultured in medium containing 1.25, 5, 7.5 or 10X NEAA produced 10-20 fold more PFC and exhibited higher survival than those cultured with no added NEAA. The omission of glutamine (Table 2) from the medium resulted in less than 5 PFC/lO”. The addition of as little as 0.5 X restored the capacity to produce PFC and increased the cell viability. Results obtained with other strains indicate that the minimum requirement is between 0.125 X and 0.25 X. Calcium ions, while not essential for the in &fro response (Table 2), maximize the capacity of cultures to form PFC. The response was inhibited over 80% by high levels (10 X ) of calcium, even though cell viability remained as high as that without calcium addition. The concentrations of components of the medium and the method of preparation are presented in Table 3. Vwiation of culture conditions. Of the techniques reported to improve the in vitro response in other culture systems (1, 13), those described in Table 4 did not significantly affect the response in the present culture medium. Cultures that were rocked on a platform at 7 rpm exhibited lower cell viability and produced fewer PFC than those held stationary. Cultures supplemented daily with medium produced only 12% more PFC than unsupplemented cultures. Since it is generally acknowledged that excessive amounts of hemoglobin in the

ANTIBODY

SYNTHESIS

TABLE PFC -~--

Glutamine

Calcium

ions

269

VITRO

2

RESPONSE IN VARIOUS CONCENTRATIONS OF NONESSENTIAL ,~JIINO ACIDS, GLCTAMINII, AND CALaunt IONS

Compound Nonessential

IN

added

Amino Acids

Concentratiw

Cell recovery

zero 1.25 s 5.0 S 7.5 N 10.0 s

31 47 47 43 SO

(%I

PFC/lO~ 400 8150 8250 8600 7500

celk ‘L (356) 6 (6850) (9700) (7800) (3700)

Zl?l-” 0.5 SC 1.0 s 2.0 s

42 64 69 62

<5 4650 4650 4.540

WI-0 1.0 s d 10.0 s

47 58 41

1170 S690 1086

a Iktermined after four days of culturing in Table 3 ex’cept for the variables shown. h Values obtained in another experiment. c 1X is 211114. d 1X is 1.26mu.

C:,BI.

spleen cells plus SRBC in medium

described

medium may have harmful effects on cells, we compared the response of spleen cell suspensions from which the mouse RBC were or were not removed (Table 4). Roth PFC and cell viability were somewhat higher in the absence of mouse RBC. The responses achieved in air ranged from SO-90% of those achieved in 10% 0, (Fig. 1 and Table 1) The lower percentage was in part a consequence of the pH of the medium at the initiation of the cultures. The pH of culture medium (in dishes) \vai varied by preincubation in : ( 1 ) a CO? atmosphere, which resulted in atmosphere (pH 7.1) ; or (3) air an acidic pH (pH 6.0-6.5) ; (2) a 5% CO,-air (pH 7.6). ‘The results indicated that the initial pH had no effect on the response of cells incubated in 10% 0, (all had 2900 PFC/lO”). If cells were cultured in air, however, an initial basic pH resulted in a lower response (1650 PFC/lO’) than that achieved when the initial pH was neutral or acidic (2410 PFC/lO”) . AntG/cpt. The in viva response of C,,Bl mice to other heterologous erythrocytes is shown in Table 5. The magnitude of the response induced by BRBC or HRBC failed to approach that induced hy SRBC. The in vitro response against SRBC, BRBC, and HRBC was consistently higher than that obtained in viva (Table 5). &cause of the high response of C,,l
270

CLICK,

HENCK,

AND

stork Ingredient

Culture

Hanks BSS Essential Amino Acids Nonessential Amino Acids NaOH Nucleic Acid Precursors Vitamins Pyruvate Glutamine PeIl~ciIIin/Streptoi71~cln

10x 50s

(1oolnM)

100s

Sterile Water The above medium is designated lx EHAA. prior to use for culture: 0. 1 11 Mercaptoethanol Fetal Calf Serum 4.4 % Sodium bicarbonate

S

0.15 * 2.5 2 2.5 L 0.25

2.5 2s 2.5s 2x 25 units or 25 ~gln/nll

100X (2OOm>1)

a Ingredients added in the order given. *After preparation, the pH of EHAA RBC storage.

10 4

5s

2 .\I 100s 100x

71.3 are added to 89 ml EH‘L-2

The following

IS

to 7 for culture

just

0.0.5 1111 10 ml 1 .6 ml

5 x 10-j hl 10%

is adjusted

100 1111

ingredient;

1\ 2x

100x

100x

ml of ,Sttrk

concentration

concentration

0

ALTER

medium

or 7.6-7.8

for

Results from the experiment shown at the bottom of Table 6 indicate that it is no longer necessary to wash RBC just prior to each experiment. After the initial washings, SRBC retained their potential to immunize in vitro cultures for up to 4 months when stored in EHAA medium at a pH of 7.6 to 7.8, even though the number of RBC declined after extended storage. In addition, the number of RBC remaining after one month of storage were found to be suitable for the plaque assay. BRBC and HRBC can also be stored for extended periods; the BRBC, however, are considered inadequate after 2 weeks of storage. Kinetics of plaque formation. Figure 3 shows the results of a representative experiment in which C&B1 spleen cells were cultured with SRBC for various periods of time. During the first 24 hr the number of PFC doubled. The maximum rate of TABLE PFC F~KMED Condition

UNDER

varied

Cultures rocked Cultures held stationaq Cultures not supplemented Cultures fed daily Mouse RBC removed Mouse RBC not removed

VARIOUS

4 CULTURE

CONDITIONS

Cel I recovery

with medium

n Determined after four days of culturing described in Table 3.

(%)

PFC/lOli

2.5 43 43 3; 46 36 CslBL spleen cells plus SIZBC

cells a

3520 4820 3925 4800 2950 2410 in 10%

O2 in tnedi~~m

ANTIBODY

SYNTHESIS

TABLE PRIMARY

RESPONSE

OF

Cs,BL

IN

271

VITRO

5

TO SHEEP,

BURRO,

AND

HORSE

RBC

PFC/lOG cells Antigen Iused for immunization

In aitro a In viva b

SRBC BRBC HRBfC None

SRBC

BRBC

HRBC

8690 280 360 290

0 780 860 0

16 42.5 2740 0

400 88 162

a Determined against indicated RBC after four days of culturing CSTBL spleen cells in 10% 02 in medium described in Table 3. * Animals were injected (I.P.) with 2-5 X lo8 SRBC, BRBC, or HRBC 4 days prior to assay against the immunizing antigen. increase, however, occurred between day 1 and 3; the doubling Thereafter, the rate declined and a maximum of 7200 PFC/lO’

time was 5-6 hr.

was achieved on day 5. Th.e cell viability declined in a linear fashion during the 6 day culture period. In unstjmulated cultures (shown by the bar in Fig. 3), 250 PFC/lOG were produced after 4 days of culture. This response has never exceeded 4-5% of that attained by ,stimulated cultures. Response of various strains. Table 7 compares the in vivo and in vitro response of three strains of mice. The results presented are from two representative in vitro experiments; similar results were obtained using mice of these strains between the ages of 2-6 months (within any experiment, all mice were within 3 weeks age of each other). A lower in vivo SRBC-response (400 PFC/106) was produced in TABLE PFC

RESPONSE

Type of SRBC used for immunization

IN VITKO

6

AGAINST

AGED

Age of RBC prior to use *

-~

AND

H-

AND

L-TYPE

SRBC

PFC/106 cells a H-type RBC

L-type RBC

L-type

Expt. :1-18 Expt. iC23

<30 days <30 days

786 4100

777 5000

H-type Expt. :1-18 Expt. ik-23

<30 days <30 days

4810 12,000

850 2900

1 day 16 days 63 days 135 davs

5100 6830 7750 7650

H-type

-

a Determined against indicated RBC after 4 days of culturing C67Bl spleen cells in medium described in Table 3. 6 SRBC stored in modified EHAA (see Materials and Methods) for stated times.

272

CLICK,

BEXCK,

AN3

ALTER

FIG. 3. Kimtics of I-‘I;C formatiou and cell viability. C,;Bl spleen cells plus SRBC were curtured in the medium described in Table 3. The bar at day 4 represents the PFC present in unprimed cultures.

Cs,B1 animals than in Balb/c (1820 PFC/106) and CBA (2050 PFC/lO”) ; in vitro however, C,,BI was the highest responder. In 10 consecutive experiments the response of C,,Bl was 5-10 fold higher than CBA and Balb/c. In fact, the ?:nvitro TABLE PRIMAKY

RESPOKSE

OF THREE

7 ~LIOCSE STRAINS

PFC/lV

TO SRBC

cells In iGtr0 b

Mouse strain in rioo a

Expt. 2213 d CBA Balb/C CuBl

2050 f 300 ( 1820 zt 6.53 400 f 1.53

2442 f 213 3610 f- 201 11.600 f 1025

Espt.

t-29 d

890 1500 4960

QAnimals were injected (I.P.) with 2-5 X lOa SRBC f our days prior to assay-. h Determined after four days of culturing spleen cells with SRBC in 10% 02 in medium described in Table 3. c Standard error. d Animals in both experiments were approximately 3 months old and their ages were within 3 weeks of each other.

APiTIKODY

SYNTHESIS

IN

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273

response of this strain always surpassed and was in many cases lo-15 fold higher than that attained in viva. Balb/c and CBA responded similarly in viva and in

vitro. DISCUSSION

Although all ingredients in the culture Culture conditions-sulfhydryl agents. medium are presumed to be important for maximum antibody synthesis. the reagent that distinguishes the present culture system is the reducing agent, MET. It is clear that the sulfhydryl group is the active moiety since three unreiated reducing agents enhanced the response. MET and DTT were the most effective reducReduced glutathione was ing agents tested, both resulting in equal enhancement. approximately SO% as effective, while L-cysteine was essentially ineffective. These findings might be expected since neither glutathione nor cysteine are strong reducing agent>. and both undergo rapid auto-oxidation. Further, the testable concentration of cysteine was limited by its precipitation from the medium at high coucentrations (which may include its optimum). Even thoug-h MET and DTT enhanced the response equally, the concentration response was lo-fold higher. This is of DTT ,required to produce a rn;ccin~u~~~ somewhat surprising since DTT is generally superior to ;LIET as a protective reagent for sulfhydryl groups during protein purification procedures ( 14). The higher requirement of DTT may, in part, be attributable to its formation of stable bidenate chelates with the metal ions present in the culture medium (15). Although it is not known how the sulfhydryl agents enhance antibody synthesis in vitro, other experiments indicate that they affect events that occur within the first 24 hr (16~). Possible modes of action include : modification of the antigenic determinants, resulting in a more powerful immunogen ; effects on components of the medium; maintenance in the reduced state of thiol groups on cell membranes (gamma globulin receptor molecules ?) ; or activation of SH-requiring enzymes. Preliminary studies indicate that they do not affect the system via the first two possibilities. The enhancement of an immune response 1)~ reducing agents described in this paper is not unique. Lymphocyte transformation has heen increased by the reducing agent, L-cysteine (17). K esults presented here suggest that a more effective stimulation may have resulted if stronger agents (RI ET and DTT > rather than cysteine had been used. In another study, MET enhanced a mixed leukocyte culture (MLC) reaction in the absence of protein supplements (IS). Culture conditions-othrr ingrcdimts. Other culture ingredients found essential to maximize the response were L-glutamine, nonessential amino acids, calcium ions, and nucleic acid precursors. The response in the absence of nonessential amino acids was small and confirms the report of hlishell and Dutton (I ) that these are required to maximize the in z~itro response. Since this response without added NEXA approximates that in viva, it might be tempting to suggest that these cuture conditions more closely reflect the in viz!0 conditions. That this lo\vered response is in fact a result of suhoptimal culture conditions is indicated by a similar lo-15 fold reduction of the in zlitro response of Ball)/c, ;1 strain whose in viz,0 response is similar to its i?t z&o response.

274

CLICK,

BENCK,

AS;D

ALTEK

A good response was attained in the absence of calcium ions and nucleic acid precursors ; however, addition of either resulted in a higher response. The suboptima1 responses attained in vitro in the absence of and at high concentrations of calcium ions are similar to the calcium-dependent depressed responses achieved in viva ( 19). The dependence of the in vitro response on calcium concentration will facilitate the assessment of its function (19) in antibody synthesis. L-Glutamine is not nutritionally essential for nitrogen balance in animals, yet some cell types have a population-dependent requirement for it in culture (20). Since the number of spleen cells seeded in the present experiments may be considered high by tissue culture standards and since mouse spleen tissue contains small amounts of glutamine synthetase (21, 22), it might be expected that such cells would function for short periods of culture without added glutamine. As demonstrated, however, no response was obtained in medium lacking this amino acid even though cell viability was high, a finding similar to that described for another culture system-the mixed-leukocyte culture reaction (23). This apparent requirement may merely reflect the restoration of glutamine lost by cellular leakage or may represent a more important role in antibody synthesis per se. Culture conditions-fechnical. The present culture system, which permits immunization of mouse spleen cells by heterologous RBC in vitro, has several technical advantages over the systems introduced by Mishell and Dutton (1) and by Marbrook (2, 3) : (1) Th e routine of feeding cultures daily has been eliminated, thereby minimizing the risk of contamination. (2) Culturing under stationary conditions in Petri dishes allows visual examination with time-lapse cinematography of cell-cell interactions in which the antigenic RBC can be identified. (3) The procedure of washing RBC prior to each experiment is no longer necessary. (4) High responses can be achieved in an air-CO, atmosphere, thus abolishing the need for expensive gas mixtures. Mqnitz~de of the in vitro response. The in vitro responses obtained in the present culture system are routinely higher than those reported for C,,Bl (13, 24-27), Balb/c (25, 28), and CBA mice (2, 8, 27-29) using other culture systems. For example, in 26 consecutive experiments in our system, the mean response of C,,Bl. was 7200 PFC/lOG on day 4 (range of 200@-12,000 PFC/lOF) in contrast to 853 PFC/lO” reported by Mishell and Dutton (24), 173 PFC/106 by Hirano and Uyeki (25), 800 PFC/106 by Hartmann (26), 900 PFC/lOG by Pierce and Benacerraf (13), and no response whatsoever reported by Tan and Gordon (27). The use of L- or H-type SRBC cannot account for the differences in magnitude since Mishell and Dutton also used H-type SRBC (24). I n addition. the responses obtained in our system with L-type RBC were as much as lo-fold higher than those reported by other investigators. The most probable explanations for the higher responses in our system are the modified culture conditions discussed. Inwzune

responses

in comnton

cztlture

conditions.

To date, of the three immune

responses that have been achieved with mouse spleen cell suspensions in vitro, only tolerance and antibody synthesis to flagellin antigens have been attained under identical culture conditions (7, 8). Since antigens can induce more than a single type Of response in an animal, similar culture environments for all types of immune responses would permit a better understanding of their illterrelationships. The

ANTIBODY

SYNTHESIS

IN

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275

present culture system has been adapted so that the genetic disparity of allogeneic or coisogeneic strains can be determined by the MLC reaction (IS), a presumed measure of cell-mediated responses. Furthermore the present culture system approximates the system used for tolerance induction (7, 8) closely enough that it could be easily adapted for tolerance studies. If so, aspects of three immune responses (tolerance, antibody synthesis, and cell-mediated) could be studied under the same ijz vitro conditions. In vivo--in vitro dijjerences. Ideally, a tissue culture environment should approximate the :in 24~0 milieu. The present findings re-emphasize that even though tissue culture systems have great potential for the analysis of biological events, caution must be exercised before concluding that in vitro findings reflect in viva events. In the two most widely used culture systems (1, 2) the “primary” in vitro antibody response closely parallels that observed in viva with respect to magnitude, kinetics, effect of antigen close, and the inhibitory effect of passive antibody. However, at least five parameters delineate the in vitro response from the in vivo activity; the first four may reflect the inability of cells in culture to switch from IgM to IgG synthesis. ( 1) The delayed decline of Igbf synthesis in vitro (1) ; (2) the heightened in vitro IgM response after in vivo priming (1) ; (3) the failure of cell cultures from unimmunized mice or mice which were eight or more weeks post-SRBC immunized to produce indirect PFC efficiently (30, 31) ; (4) the suppression of only the ~PZvitro response of eight week post-immunized mice by anti-SRBC antibody (30), ; and (5) the discrimination of SRBC determinants in vitro (1). The results in this paper further distinguish the in vitro from the in vivo responses. The magnitude of the “primary” in vitro response did not always parallel the in viva response ; the correlation was strain dependent. The in vitro responses of Balb/c and CBA approximated their respective in vivo responses, whereas C,,Bl clearly responded higher in vitro than in viva. In fact, even though a large variation in magnitude has been obtained in different experiments with this strain, in no case has the in vitro response closely approximated the in vivo response. Genetic control of the response. Comparison of the in vitro responses of three strains investigated showed that C&B1 was the highest responder, Balb/c was intermediate, and CBA was the lowest. However, the in vivo responses of these same strains ranked in inverse order. These results suggest the loss or impairment in vitro of some mechanism that regulates the extent of antibody synthesis in z&o. Since this mechanism is under genetic control, it appears to be different from that described by Mishell and Dutton (I), i.e., the inability to terminate the increase of 19 S PFC in vitro at the time it would have terminated in viva. Su~~~zzation. A culture system for cell suspensions from mouse spleens which differs from those currently in use has been described. The present system offers technical advantages which simplify the culture procedure. In addition, with simple variation, the present system offers the possibility of a common culture medium which w011lc1 facilitate the study of the interrelationship of tolerance, cell-mediated responses and antibody synthesis. The magnitude of the responses of spleen cells cultured in this system was markedly greater than those reported using other culture systems. Furthermore, it was observed that the ilz vitro responses did not always parallel the in viva responses as

avitrO previously reported ; the relationship was strain dependeilt. This ip~ z&.-in dichotom\- provides an important additional tool for the study- of the molecdar and cellular events of antibody synthesis. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

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