Mechanisms of antibody formation

Mechanisms of antibody formation

CELLULAR IMMUNOLOGY 20, 42-53 (1975) Mechanisms II. Use of DNP-Ficoll P. R. B. MCMASTER, of Antibody Formation in Studies of Hapten-Specific ...

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CELLULAR

IMMUNOLOGY

20,

42-53 (1975)

Mechanisms II. Use of DNP-Ficoll

P. R. B. MCMASTER,

of Antibody

Formation

in Studies of Hapten-Specific

R. J. BACHVAROFF,

AND

B-Cell

Responses1

F. T. RAPAPORT *

DepartrPlent of Surgery and the Institute of Reconstrucfive Plastic Surgery, New York University Medical Center, New York, NY 10016; the Laboratory of Immunology, National In&t&e of Allergy and Infectious Diseases, Bethesda, Maryland 20014; and the Research Service, Manhattan Veterans Administration Hospital, New York, NY 10010 Received April

18,197s

Mice injected with 10 pg Ne-dinitrophenyl-L-lysine-Ficoll (DNP-F) preparations ranging widely in epitope density (DNP 52-F to DNP 11.5-F) generated longlasting (6 months) anti-TNP IgM and IgG responses, as measured by the number of spleen PFC produced against sRBC-TNP or hRBC-TNP. The immunogenicity of DNP-F correlated directly with the epitope density; DNP 1.5-F was nonimmunogenic. When mice immunized with DNP-F and actively producing antibodies were challenged with 2 mg DNP-F at different time intervals after sensitization, a rapid and longlasting reduction in the formation of anti-TNP IgM and IgG PFC occurred. Mice were primed in viva with sRBC, and spleen cultures were initiated in the presence of sRBC-TNP. Addition of a large dose of DNP-F at the start of the cultures or at 1 and 2 days later greatly reduced but did not completely eradicate the formation of anti-TNP IgM PFC. The production of anti-sRBC PFC was not affected. This specific tolerogenic effect persisted when the cells were washed after a sui:able period of exposure to DNP-F, and were then recultured. Generally, exposure of the cells to DNP-F for 8 hr sufficed to reduce the number of TNP PFC observed on subsequent days; exposure for 2 hr was inadequate. These observations indicate that DNP-F does not have to be continuously present in the culture medium to exert its tolerogenic effects. The concentrations of DNP-F capable of exerting tolerlogenic effects do not act by killing the TNP-PFC. Rather, this effect appears to have been a consequence of the restriction of clonal expansion of hapten-specific B-cells by DNP-F. The results also suggest that DNP-F may exert a tolerogenic effect after clonal expansion. In this instance, antigen-antibody complexes may have formed on the surface of hapten-specific B cells, with deamplification of antibody gene transcription.

INTRODUCTION The early work of Landsteiner (1, 2) has demonstrated that antibody responses to certain small chemical determinant groups can be induced if such groups are coupled to a suitable large carrier molecule. Such small chemical determinants, or haptens, have been used extensively since then in studies of the immune response (3). It has been observed in the course of these studis that the nature of the carrier (4) plays a vital role in the excitation of thymus-derived cells (T cells) in T-cell 1 Presented in part at the 1974 Meeting of the American Association of Immunologists. 2 Irma T. Hirsch1 Career Scientist Award. 42

CopyrightQ 1975by AcademicPress,Inc. rightsof reproductionin my form reserved.

All

HAPTEN-SPECIFIC

B-CELL

RESPONSES

43

dependent antibody responses, and for the stimulation of bone marrow-derived cells, or B cells (5) in T-cell independent responses. Humoral responses to varied and complex protein antigens, such as bovine gamma globulin or albumin, for example, appear to require the presence of T cells to stimulate antibody formation (6, 7). In contrast, more uniform repetitive carrier molecules such as pneumococcal polysaccharide, for example, appear to have the capacity to activate B cells directly. without any participation by T cells (S-10). These polysaccharicles occur generally in two forms, either as a linear chain or as branched molecules. Certain aspects of the mechanisms of antibody formation to T-cell dependent immunogens have been described in an earlier report ( 11). The present study is concerned with mechanisms of humoral responsiveness to T-cell independent immunogens, utilizing haptens coupled to branched, repetitive polysaccharide molecules as the antigenic prototype. For this purpose, a sucrose polymer, Ficoll. was coupled with the hapten NC-dinitrophenyl-L-lysine (DNP), yielding DNPFicoll complexes in which the DNP substitution ratio ranged from 1.5 to 72 DNP per Ficoll molecule. The resulting materials were assessed with regard to the comparative efficacy of different hapten-carrier molecule substitution ratios as immunogens and tolerogens. The results indicate that dinitrophenyl-substituted Ficoll (DNP-F) preparations with substitution ratios ranging from DNP 11.5 to DNP 32 are active as potent immunogens. In addition, larger doses of DNP-F were found to have profound tolerogenic properties in Go and in vitro. Taken together, the data presented in this report highlight the potential usefulness of DNP-F preparations in the further elucidation of mechanisms of antibody formation and immunological unresponsiveness to T-cell independent antigens, MATERIALS

AND METHODS

AnimnZs. Three to six month old C57B16mice were used throughout merits; the animals were maintained on a standard Purina pellet diet.

the experi-

Antipns DNP-Ficoll. 1.9 g of the sucrose polymer Ficoll (Pharmacia Fine Chemicals? lot 2300, MW 400,000 d) were suspended in 4.0 ml of distilled water, and 4.0 ml 1 N NzOH with 0.50 g of KHC03 were added. As soon as the components dissolved, a freshly prepared suspension of 2oo-600 mg of cyanuric chloride in 2 ml dimethylformamide was added, and the mixture was stirred for 2-4 min at room temperature. One gram of NC-dinitrophenyl-L-lysine (DNPL) in 1.0 ml of distilled water, pH 9.0 (adjusted with NaOH), was then added. This mixture was stirred overnight, and was dialyzed against distilled water for 2 weeks; it was then dialyzed for 3 days against isotonic saline solution (the dialysis bags were repeatedly boiled in a solution of Na2C03 and EDTA before use). The preparation was centrifuged at ZOOOg, and the supernatant was passed through a 0.22 ppore-size filter. The concentration of DNP in the final preparation was determined spectrophotometrically at 365 nm, using a molar extinction coefficient of 16,400. The carbohydrate content was determined by the phenol sulfuric acid test (12). Sheep (s) and horse (h) red blood cells (RBC) (Animal Blood Center, Syracuse, New York) were washed three times in balanced salt solution (BSS)

44

MCMASTER,

BACHVAROFF

AND

RAPAPORT

(13), and were used as antigens. The washed cell suspensions were adjusted to the desired concentration in standard fashion (14). Erythrocytes were lightly and heavily substituted with TNP by the procedures of Rittenberg and Pratt (15) and Kettman and Dutton (16), respectively. RBC were substituted with dinitrophenyl as described by Trump (17). Tissue cultures. Spleen cell cultures were initiated and maintained as described by Mishell and Dutton (13), except that 1O-5 A4 Z-mercaptoethanol (18) was added to the culture medium. Usually, 1.5-2.0 X lo7 spleen cells per culture were challenged with 4.0 X lo6 with fresh sRBC heavily substituted with TNP. The generation of IgM-producing cells in each culture was assayed by enumerating the direct PFC, utilizing the plaque technique of Jerne, Nordin, and Henry (19), as modified by Mishell and Dutton (13). Where anti-TNP antibody responses were measured, lightly substituted hRBC-TNP were used for plaquing. Guinea pig serum absorbed three times with RBC was used as the source of complement in the plaquing technique. The IgG anti-TNP response was measured by enumerating the indirect PFC as described by Pierce et al. (20). Rabbit anti-mouse IgM and anti-IgG sera were kindly provided by Dr. Asofsky. The number of PFC counted each day which is listed in the reported results represents average counts from three simultaneously prepared culture dishes plaqued in triplicate. The number of PFC formed in response to DNP-RBC or TNP-RBC was regularly the same in our hands, but, since TNP-RBC are more stable than DNPRBC, they were generally used instead of DNP-RBC for plaque assays.

RESULTS In Vivo Ivnmunogenic Activity

of DNP-Ficoll

Different sets of mice received 10 pg of DNP-F in which the number of DNP groups per Ficoll molecule was 32, 25.7, 11.5, or 1.5, respectively. Beginning with the first day after immunization, four from each experimental group were sacrificed daily ; spleen cell suspensions were prepared from each animal and the number of direct PFC against TNP-sRBC was determined. As shown in Fig. 1, all mice given DNP-F with substitution ratios of DNP 32, 25.7, and 11.5 generated antiTNP PFC; the magnitude of the response generally diminished with reductions in the substitution ratio; there was no detectable response to DNP 1.5-F. When the dose of DNP 1.5-F was increased to 350 rg, only a barely detectable number of anti-TNP PFC appeared (73 2 15 PFC on day 4)) in contrast to the strong response obtained with as little as 0.3 pg of DNP 32-F (616 * 41 PFC on day 4). If 50 pg of Vinblastine was given intraperitoneally on days 1, 2, 3, and 4 to mice injected intravenously with DNP-F with substitution ratios of 11.5 to 32, no PFC were formed during the next few days. This result indicates that the plaques observed each day were formed by cells that had recently undergone division. Table 1 illustrates the results of repeated injections of DNP-F. One group was injected intravenously with 10 pg DNP 25.7-F; a second received 10 pg Ficoll without DNP, and a third group was left untreated. Three days later, all mice received 10 pg DNP 25.7-F intravenously. The response of mice injected twice with DNP-F was similar to that of mice injected just once with this material. In these experiments, it was observed that one-third of the mice injected with 100 pg

HAPTEN-SPECIFKB-CELLRESPONSES

Oi

45

12345678 DAYS AFTER INJECTION DNP- FICOLL

OF

FIG. 1. In viva immunogenic activity of DNP-Ficoll. Mice were injected with 10 pg DNP-F preparations containing 32, 25.7, 11.5, and 1.5 DNP groups per Ficoll molecule and the generation of anti-DNP IgM PFC was measured utilizing sRBC-TXP for plaquing.

Ficoll and subsequently with 10 pg DNP-F died within 3 days after the second injection. Figure 2 compares the IgM and IgG anti-TNP responses observed in mice given 10 pg of DNP 25.7-F. IgM a.nd IgG PFC first appeared on the third day after injection; both rose to a peak on day 5, diminished gradually until the eighth day, and then remained relatively constant in number for as long as 6 mos. In Vivo and In Vitro Toleroyenic Activity

of DNP-Ficoll

Mice immunized with 10 PLgof DNP 25.7 intravenously were reinoculated by the same route with 2 mg of the same preparation, given 5, 6, or 30 days later. As shown in Fig. 2, there was a marked diminution in the number of TNP PFC within 24 hr after the second injection; this diminution persisted for the entire duration of the study (30 days). If immunized mice were given Ficoll alone, there was no effect on the ability of the animals to form TXP-PFC. In further studies, mice were injected with 2.0 x 10: sRBC; 4 days later (da! 0), their spleen cells were cultured and stimulated with sRBC-TNP. The antiTNP (utilizing hRBC-TKP) and anti-sRBC responses observed in such mice were compared with the results obtained in cultures to which 350 pg of DKP 30-F had been added on culture days 0, 1, 2, 3, or 4. As noted in Table 2, addition of this dose of DNP-F to the cultures greatly reduced the anti-TNP response. without affecting the anti-sRBC response. The addition of comparable amounts of Ficoll had no measurable effects on either response. The ability of DNP 30-F to inhibit in vitro responses to TNP was dose-dependent. As noted in Table 3, spleen cell cultures obtained 4 days after injection of sKBC into mice were challenged in vitro with sRBC-TNP, and graded amounts of DNP 30-F were added to each culture. The efficacy of DNP 30-F in suppressing anti-TNP responses

46

MCMASTER,

v3

4

BACHVAROFF

5

6

7 0

AND

2030

DAYS AFTER INJECTION

RAPAPORT

60

100

OF DNP-FICOLL

Mice were injected with 10 pg DNP FIG. 2. In viva tolerogenic activity of DNP-Ficoll. 25.7 and the anti-TNP IgM (solid line) and IgG (broken line) responses were followed. On the day indicated by an arrow, lo-15 mice were given 2 mg DNP 25.7-F and the responses were measured the next day, and at various time intervals thereafter.

decreased in direct proportion to the dose used, and was absent when less than 0.37 pg of DNP 30-F was used. Table 4 provides an assessment of the tolerogenic activity of DNP-F preparations bearing different amounts of DNP per Ficoll molecule. When DNP 2.5.7-F, DNP 11.5-F, and DNP 1.5-F were used in vitro as described above, preparations containing 2.5, 5.0, or 1.0 rg of DNP on Ficoll caused uniform reductions in the number of anti-TNP PFC. Lower amounts of DNP were not effective. (It is important to note, however, that different batches of DNP-F with the same DNP substitution ratios often produced variable suppressive effects ; no explanation of this phenomenon is available at present). The effect of removing DNP-F from the cultures was also studied. For this purpose, DNP 30-F was added to the cultures at suitable times and then, 24 hr later, the culture dishes were scraped with a rubber policeman, the cells were collected; they were washed twice in BSS and were then recultured in fresh medium z&ho& DNP-F. As shown in Table 5, the presence of 375 pg of DNP 30-F/ml at O-24 hr, 24-48 hr, or 48-72 hr after initiation of culture decreased the number of anti-TNP-PFC present on days 3, 4, and 5, respectively. Incubating spleen cells with DNP 30-F for 2 hr at 37°C did not, however, have any effect upon the formation of anti-TNP PFC ; the minimum time required to effectively reduce the number of anti-TNP-PFC was 6-8 hr. In further studies of the tolerogenic activity of DNP-F, a group of mice was injected intravenously with 2 mg of DNP 25-F, and a second group received 10 pg DNP 25-F. Simultaneously with DNP-F, the animals in both groups received 2.0 x lo7 sRBC. A third group of mice was only given sRBC. Four days later, spleen cell cultures from all three groups of mice were initiated in the presence of sRBC-TNP. As shown in Table 6, cultures initiated from mice which had been treated with 2 mg of DNP 25-F and sRBC produced very few anti-TNP PFC, as compared

to cultures

initiated

from mice injected with

from

mice given

10 pg of DNP

sRBC

alone.

Cultures

prepared

25-F and sRBC produced a markedly

increased number of anti-TNP PFC. Cultures comparable numbers of anti-sRBC PFC.

from

all three

groups

produced

--

-.-~ ~__

10 pg Ficoll 10 pg DNP 25.7 Ficoll Nothing

Day 0

Mice injected

In

__. 25.7 Ficoll 25.7 Ficoll 25.7 Ficoll

Day 3

1

40 f 45 f 10 f

10 12 3

Day 3

AFTER Two

TABLE

Viz10 IMMUNE RESPONSE

10 pg DNP 10 pg DNP 10 pg DNP

with

THE

INJECTIONS

732 f 659 f 846 f

51 63 48

Day 4 395 f 474 f 662 f

41 47 51

Day 5

280&31 255 f 41 495 f 38

Day 6

Anti-hRBC-TNP IgM PFC per lo8 cells found after the first injection of DNP-Ficoll on

DANP-Fko~~

218 f 211 f 421 f

21 32 41

Day 7

E g

i E ; z Y 1 F

$

X %

48

MCMASTER,

BACHVAROFF

AND

TABLE EFFECT OF 35Opg DNP

RAPAPORT

2

30 FICOLL ADDED TO CULTURES ON DIFFERENT

DAYSO

IgM PFC per lo6 cells DNP-Ficoll added

Day 3 sRBC

o-5 1-5 2-5 3-5 4-5 None

7572 f 6643 f 6626 f 8103 f

0 Mice injected

300 351 278

411

Day 4

hRBC-TNP

sRBC

58 f 75 f 7oi 3.50 f

4375 f 189 4000&211 3612 f 203 4509 zk 301 4180 f 32

1.5 17

30

with sRBC on Day

-4;

cultures

Day 5

hRBC-TNP 4Oill 48 i 49 f 118 f 439 i

stimulated

sRBC 1802 1901 1520 2518 1162 1463

13 16 22 37

with sRBC-TNP

f f f f f i

hRBC-TNP 2.51 188 211 311 321 271

5&l 76fll 21 f6 39 + 9 23 f 5 259 f 25

on Day 0.

A final question concerned with viability of TNP PFC incubated with a high dose of DNP-Ficoll. In order to test this variable, spleen cells actively producing anti-TNP antibodies were plaqued, and 50 plaques were circled with a black marker. The plates were overlaid with DNP 72 Ficoll and were incubated for an additional 6 hr at 37°C. At the end of this period, the DNP-F was removed and 1% Eosin solution was placed over the agar layer. The nucleated cells present in the center of each plaque were examined microscopically ; none of the PFC studied was stained by Eosin. DISCUSSION First synthesized approximately 3 yr ago, DNP-Ficoll preparations have been of significant value in studies of mechanisms of antibody formation. The extensive studies of Paul et al. (21) and of Mosier et al. (22) have provided considerable information on the antigenic properties of DNP-F. The recent observation that DNP-F also has tolerogenic properties (23) has added further interest to the use of such preparations in studying mechanisms of immunological responsiveness. Results of the present study indicate that the immunogenicity of DNP-F is a function of the DNP substitution ratio of each preparation. At the lowest substiTABLE In

Vitro

EFFECT OF DIFFERENT AMOUNTS OF DNP 30 FICOLL UPON THE DEVELOPMENT OF PFC TO hRBC-TNP*

Amount of DNP 30 Ficoll added per culture at 0 time None 372.0 pg 37.2 fig 3.7 rg 0.37 /.lg a Mice injected

3

pg DNP on Ficoll per culture 0 11.2 1.1 0.11 0.01

day -4

with sRBC ; cultures

Anti-hRBC-TNP Day 3 244 40 100 104 181

f 31 f 8 f 1.5 f 18 f 21

stimulated

IgM PFC per lo6 cells Day 4 410 f 58 f 134 f 174 f 3.58 f

25 7 17 18 28

with sRBC-TNP.

Day 5 127 f 13 5fl 49 f 7 51 f5 176 f 11

HAPTEN-SPECIFIC

B-CELL

TABLE In

Vitro

4

EFFECT OF DNP FICOLL OF DIFFERENT SUBSTITUTION UPON THE DEVELOPMEKT OF hRBC-TNP PFCU

Type of DNP Ficoll added

rg DNP on Ficoll per culture

None DNP 25.7 DNP 11.5 DNP 1.5 a Mice injected

49

RESPONSES

Anti-hRBC-TNP

0 3.5 5.0 1.0 day -4

with sRBC;

cultures

RATIOS

Ig>‘I PFC per 10” cells

Day 3

Day 5

Day 5

882 i 51 13 f 2 5*1 9fl

327 A 18 Lfl 19 zt 3 13 3z 2

84 f 11 0 7fZ 20 f 3

stimulated

with sRBC-TSP.

tution ratio tested (DNP 1.5), DNP-F lacks detectable activity, regardless of the total dose of DNP injected. Evidence is also presented that the antibody response to DNP is remarkably long-lasting when induced with DNP-F, and that the formation of IgM and IgG anti-hapten PFC may continue for as long as 6 mo after a single intravenous injection of DNP-F. The prolonged duration of this response may be due to the persistence of DNP-F at sites of antibody formation, in a manner similar to that described for pneumococcal polysaccharide III (24). It is important to note that the tolerogenic dose of DNP-F did not eliminate all cells capable of producing TNP PFC. As Paul et al. (21) have noted, this ma) indicate that cells making low affinity antibody may escape the tolerogenic effect, for reasons that remain obscure at present. The absence of an amnestic response to DNP in animals primed with an immunogenic dose of DNP-F or Ficoll alone (Table l), supports the conclusion of Paul ef al. (21) and of Mosier et al. (22) that DNP-F is a “T-independent” B cell stimulator. It is of interest in this regard, however, that the injection of mice with immunogenic doses of DNP-F and sRBC 4 days prior to the initiation of spleen cell cultures in the presence of sRBC-TNP causes a significant increase in the response of such cultures to TNP, when compared to cultures prepared from mice injected with sRBC alone or with DNP-F alone (Table 4) This result is compatible with the notion that DNP-F stimulated B cells may become Inore TABLE TMM~XOS~PPKESSI~E

EFFECT

OF DNP

Time DNP 30 Ficoll added/372 pg per culture

Time of cell washing h-1

0 None added 24 hr None added 48 hr None added

24 24 48 48 72 72

0 Mice injected

day -4 with sRBC;

30 FICOLI.

5 PKESENT

IX THE

Anti-hRBC-TSP Day 3 54 242 32 155 22 197 cultures

f rt f zk f +

7 21 5 11 3 18

stimulated

CULTUHBS

FOR 2-l HOC-KYI

IgM PFC per 106 cells Day -1 30 439 21 249 21 351

zk rt xt zk rt f

6 32 5 22 1 21

Day 5 38 259 20 146 20 221

Day 0 with sRBC-TNI’.

f zt f f f f

6 28 4 15 1 25

0 Cultures

8835 f

sRBC

8571 f 2fl 9125 f

with sRBC-TNP.

6

125

312

211

221

3100 i 167 f 458 f

2fl

27 f

110 21 33

3

hRBC-TNP

sRBC

IgM

INJECTED WITH

195

221

2718 f 70 f 1021 f

173 f

72 f

75 6 101

15

8

hRBC-TNP

Day 4

721 f 27 f 628 f

525 f

61.5 i

sRBC

61 3 31

33

41

652 f 46 f 496 f

37 f

5fl

39 7 30

3

hRBC-TNP

Day 5

PFC per 10s cells found on days of culturing

5121 & 121 70 f 9 1828 f 58

4397 f

5967 f

of sRBC and hRBC-TNP

Day 3

Number

7346 f

stimulated

TABLE

RESPONSE -0 TNP OF SPLEEN CULTURES PREPARED FROM MICE IMMUNOGENIC OR IMMUNOSUPPRESSIVE DOSES OF DNP-FICOLLO

10 rg DNP 21 F co11 and 107 sRBC 10 fig DNP 21 Ficoll 107 sRBC

DNP 52.7 Ficoll mg and 107 sRBC

with

Vitro

2 mg DNP 21 Ficoll and 107 sRBC

2

initiation

Mice injected 4 days prior to culture

THE In

!a 4;d

HAPTEN-SPECIFIC

B-CELL

RESPONSES

51

sensitive to stimulatory T cells involved in the mediation of responses to sRBC. Alternatively, DNP-F may have increased the response of T cells to subsequent exposure to sRBC-TNP. The results of the present study have provided evidence that DNP-F is not onl) an effective immunogen, but also a remarkably potent tolerogen. Administration of a high dose of DNP-F during the induction of an anti-DNP-F response in viva, or after stabilization of such a response has been shown to profoundly decrease the number of anti-TNP IgM and IgG PFC measured 24 hr later. This state of tolerance has persisted for at least 30 days after a single injection of a tolerogenic dose of DNP-F. Similar long-lasting paralysis for pneumococcal polysaccharide III was first observed by Felton et al. (25). The long-term duration of this effect is currently under study. Earlier studies of Diener and Armstrong (26) have demonstrated the value of in vitro systems for the elucidation of mechanisms of immunological tolerance. These authors observed that polymerized flagellin can act as a tolerogen in tissue culture, and that this phenomenon involved an active metabolic process. More recently, Rittenberg and Bullock (27) and Feldmann (28) have shown similar properties for haptens coupled to proteins. In the latter studies, Feldmann (28) demonstrated that DNP coupled to polymerized flagellin (POL) can induce antibody formation or tolerance, and noted that DNP-POT> preparations with a low epitope density of DNP molecules per monomeric unit of flagellin can induce antibody formation but not tolerance. If there were three DT\‘P groups per monomeric subunit, either immunity or tolerance could result, depending upon the concentration of DNP-POL in the culture. Higher epitope densities (DNP > 3) did not stimulate antibody formation but readily induced tolerance (26). When DNP-F was used in the present study, however, all of the DNP-F preparations used, including the DNP-F with a DNP 1.5 substitution ratio per carrier molecule. had tolerogenic effects. The quantity of each DNP-F preparation needed to induce tolerance in vitro also appeared to be related to the total amount of DKP, rather than to the number of carrier molecules bearing one or more DNP groups. It is worthy of note, however, that certain batches of DNP-F with the same hapten substitution ratio required considerably more material than others to induce tolerance. The time required for the induction of tolerance in vitro also appeared to be a function of the number of DNP groups present per molecule of carrier. Preparations with the lowest epitope densities (DNP 1.4 to DNP 30-F) required 6-8 hr to induce tolerance; exposure of cultures to such preparations for shorter times had no apparent effect. Two other preparations, DNP 50 and DNP 71 had the capacity to induce tolerance more rapidly (unpublished observations). It is not yet clear, however, if the ability to induce tolerance more rapidly was determined by the epitope density or if it was a result of some other as yet unknown characteristic of the DNP-F preparations used in this particular case, Feldmann (28) has observed that DNP-lysine can compete effectively with the immunogenic or tolerogenic properties of DNP-POL. Upon removal of this competitor and replacement of tolerogenic DNP-POL molecules, their effectiveness was restored. In order to test the possibility that DNP-F might simply exert its effects by inhibiting the immunogenic action of TNP-sRBC, tolerogenic DNP-F was added to the appropriate cultures, and was then washed off. As noted in Table

52

MCMASTER,

BACHVAROFF

AND

RAPAPORT

5, there was still a pronounced reduction in the number of anti-hapten PFC produced. The only requirement was that the cells must be left in contact with tolerogenic DNP-F for a sufficient period of time (6 hr). It is also of interest that DNP-F exerted its tolerogenic effects if added at the start of the culture, as well as when added after the cells had begun to produce antibody. Also, the higher the substitution ratios of DNP per Ficoll molecule, the shorter was the exposure time needed to suppress responses to the hapten. The system used in experiments where spleen cells from carrier-sRBC-injected mice are challenged in vitro with sRBC-TNP requires the collaboration of T and B cells for the generation of high-specificity anti-hapten PFC (4, 5). This system has the advantage of providing a separate measure of the response to carrier sRBC and to the hapten. In all instances where a tolerogenic dose of DNP-F was given to cultures, the antibody responses to sRBC alone remained as high as those seen in the untreated cultures. These results indicate that DNP-F did not affect the function of sRBC-specific T or B cells. Rather, the tolerogenic effect of DNP-F appears to have been exerted solely upon hapten-specific B cells. This conclusion is supported further by the observation that when mice were injected first with carrier-sRBC and a tolerogenic dose of DNP-F, the subsequent in vitro response to TNP was greatly reduced in those cultures challenged with sRBC-TNP. Indeed this result indicates that even in vitro T-cell helper effects failed to alter the tolerant state of hapten-specific B cells. A number of mechanisms have been proposed for the induction of immunological tolerance (29). The results of the present study provide evidence that the tolerogenic effects of DNP-F in vitro are not a consequence of anti-TNP PFC death. The suppression of anti-TNP PFC observed when DNP-F is added simultaneously in vitro with the sRBC-TNP immunogen may, rather, be a consequence of the restriction of clonal expansion of hapten-specific B cells by DNP-F (29). The tolerogenic effect of DNP-F after clonal expansion may be the result of formation of antigen-antibody complexes on the surface of hapten-specific B cells, with subsequent deamplification of antibody gene transcription. ACKNOWLEDGMENTS The excellence of the technical assistance of Irene Allan, Susan Ball, Louis Browne, and Lilling Wei is greatly appreciated. Supported by a Grant from The John A. Hartford Foundation, Inc. ; supported in part by Grants GM-12748-11 and AM-14059-15, National Institutes of Health, Bethesda, Md.

REFERENCES 1. Landsteiner, K., and Lampl, H., Biochem. Z&t. 86, 343, 1918. 2. Landsteiner, K., “The Specificity of Serological Reactions.” Harvard University Press, Cambridge, 1945. 3. Goebel, W. F., and Avery, 0. T., J. Exp. Med. 50, 521, 1929. 4. Katz, D. H., Paul, W. E., Goidl, A., and Benacerraf, B., J. Exp. Med. 132, 261, 1970. 5. Claman, H. N., Chaperon, E. A., and Triplett, R. F., Proc. Sot. Exp. Biol. Med.. 122, 1167, 1966. 6. Taylor, R. B., Nature (London) 220, 611, 1968. 7. Chiller, J. M., Habicht, G. S., and Weigle, W. O., Proc. Nat. Acad. Sci. USA 65, 551, 1970. 8. Miranda, J. J., Immunology 23, 829, 1972. 9. Miranda, J. J., Zola, H., and Howard, J. G., Immunology 23, 843, 1972.

HAPTEN-SPECIFIC

B-CELL

RESPONSES

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