Immunology 1976 31 541

Clonal dominance and the preservation of clonal memory cells mediated by antigen-antibody

BRIGITTE A. ASKONAS, A. J. McMICHAEL* & M. ESTELLA ROUXt National Institute for Medical Research, Mill Hill, London

Received 27 February 1976; accepted for publication

Summary. Selected B-cell clones and their well characterized monoclonal antibody products were used to analyse the role of antibody in clonal dominance and the regulation of memory cell supplies. The experimental design was to permit contact between spleen cells and antigen in vitro, and to administer antibody to DNP prior to or following cell transfers into irradiated recipients. The anti-hapten response was strongly suppressed by the clone's own antibody or higher affinity antibody administered on day 0. Antigen-antibody inhibited memory cell generation. The suppressive effect was temporary, and reversible with time and further antigen and the same clone could be induced to produce antibody again, analysed by isoelectric focusing. We were therefore not dealing with clonal deletion. Change in the source of clonal anti-hapten excluded possible effects of antibody to carrier protein or idiotypic determinants in this system. The timing of antibody administration indicates that clones already triggered in the first 4 days after antigen contact could not be suppressed by antibody. Passive antibody to DNP only suppressed when both B and T cells had been permitted contact with

March 1976

hapten-carrier protein. Alteration of the carrier protein enabled us to study the effect of antigen-antibody on B and T cells separately. B cells binding antigen and antibody to hapten were trigger ed more efficiently by fresh T cells recognizing the carrier protein than after antigen uptake alone. Antibody to DNP suppressed only when both B and T cells had taken up hapten-protein, suggesting that antigen-antibody acts centrally at the level of both B memory cells and T helper cells. This reversible antigen-antibody blockade appears to favour the preservation of a pool of long-lived memory cells rather than the priming of new clones developing from short lived precursor cells; clonal dominance ensues. INTRODUCTION The development of analytic isoelectric focusing (TEF) (Awdeh, Williamson & Askonas, 1968; Williamson, 1971) has given us a tool to follow clonal antibody production. Observations made both in intact animals and during the propagation of single antibody-forming clones in mice pointed to two phenomena, which appear to be interconnected. (1) Clonal antibody formation can continue over periods of many months in mice or rabbits (Haber, 1971) upon continued antigenic stimulation. B-cell clones do not easily exhaust themselves by maturation into immunoglobulin-secreting cells, and a

* Present address: Department of Immunology, Stanford University Medical Centre, Stanford, California 94305, U.S.A. t Present address: Department of Pathology, New York

University, 550 First Avenue, New York, N.Y. 10016, U.S.A. Correspondence: Dr Brigitte A. Askonas, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. C

11

541

542

Brigitte A. Askonas, A. J. McMichael & M. Estella Roux

supply of memory cells is maintained for a large number of cell generations (Askonas & Williamson, 1972a; Williamson & Askonas, 1972). (2) Clones once selected can dominate a response to the virtual exclusion of other clones forming antibody to the same antigen. Such clonal dominance occurs in a number of situations. A striking example comes from the serial passage into irradiated mice of single B-cell clones forming antibody to the dinitrophenyl (DNP) group. The anti-DNP response remains monoclonal, despite the presence of other precursor cells with a potential to form different anti-DNP molecules in admixed spleen cells from normal mice or mice primed with carrier protein. This 'dominance' is broken once the proliferative capacity and antibody production by the clone declines, and antigenic stimulation then results in a highly heterogenous antibody response to DNP (Askonas & Williamson, 1972b). Domination of the response by single antibodyforming cell clones can also be induced in vivo by hyperimmunizing rabbits or mice over long periods of time with bacterial vaccines or other antigens. Once clones are selected, they can produce antibody with the same isoelectric focusing pattern or idiotypic determinants for 2-3 years; this has been observed for example with carbohydrate of Salmonella (Oudin & Michel, 1969; Bordenave & Askonas, 1974), pneumococci or streptococci (Haber 1971; Krause, 1970; Braun & Jaton, 1974) and in selected restricted responses to f8-azobenzenearsonate (Winfield, Mage & Alexander, 1973). Changes in clonal patterns can occur, particularly early during the immune response, and in some immunized animals there is some selection on the basis of increasing affinity of the clonal antibodies (Kimball, 1972). We wished to find out whether preservation of memory cells and clonal dominance were mediated by antibody. Antibody-mediated suppression of immune responses has been widely reviewed (e.g. Uhr & Moller, 1968; Playfair, 1974; Werblin & Siskind, 1972), but the mechanism of action of such suppression is ill understood. In general, IgG antibody administered in tvivo with antigen can effectively suppress a primary antibody response, whereas suppression of a secondary response is harder to achieve. Suppressive antibody can be directed to the hapten or the carrier (Haughton & Makela, 1973). Whole immunoglobulin is more efficient than Fab fragments implying a role for Fc in the suppression

(Chan & Sinclair, 1971). On the other hand, certain classes and doses of antibody also have been shown to enhance antibody production, for example, IgM (Henry & Jerne, 1968) and antibody to carrier protein (Playfair, 1974). In general, heterogenous antibodies have been used in these studies, without assessment of the relative proportions of antigen and antibody. This may explain the inconsistencies and makes analysis of the underlying mechanism difficult; it has been suggested that passive high affinity antibody suppresses by competing with B-cell receptors of lower affinity or by removing antigen (Uhr & Moller, 1968), but this does not appear to be the whole explanation. In the experiments described here, we used wellcharacterized cell clones forming anti-DNP to study the effect of their own antibody or of higher or lower affinity antibody to the hapten, also monoclonal. By changing carrier protein, we had a system to study interaction with B or T cells separately. A model to explain our findings is proposed. MATERIALS AND METHODS Mice

CBA/H mice were maintained as an inbred line at the National Institute for Medical Research and used between 3 and 8 months of age. Selection and propagation of cell clones forming anti-DNP Single antibody-forming cell clones were selected as previously described (Askonas et al., 1972; McMichael & Willcox, 1975) and several clones, expanded to produce high levels of antibody (partly with very high affinity) were passaged i.v. into irradiated syngeneic recipients with 1-10 ug antigen in saline and serum antibody was collected. In general 3-8 x 106 spleen cells were transferred and to avoid limiting numbers of T cells, 2-4 x 106 carrier-primed cells were regularly administered simultaneously. Antibody suppression experiments Clonal and carrier primed spleen cells were suspended in Eagle's minimum essential medium (MEM) in 10% foetal calf serum (FCS), penicillin and streptomycin, and washed once in medium. 2 x 107 cells/ml were then incubated at + 40 or 20° for 1 h in the above medium (excluding FCS), with antigen as indicated (10 pg/ml DNP-bovine gamma-

Clonal memory cells globulin (BGG), 5 ug/ml DNP-Maia Squinado haemocyanin (MSH) or 50 pg/ml DNP-ovalbumin (OA). The incubation volume was diluted, the cells pelleted for 8 min at 250 g and washed once with medium. The cells were then transferred i.v. into irradiated (660 r) CBA/H mice, 9-11 days later, serum antibody was titered and characterized by isoelectric focusing. Recipient mice were boosted as indicated and then serum analysed again for antiDNP 9-11 days later. Passive monoclonal antibody of known titre was administered i.p. 1 h before cell transfers or 2-4 days after the cell transfer as indicated. Antibody equivalent to an ABC of 1-5-2 was generally administered (of the order of 5-10 pug according to its affinity). DNP-proteins These were prepared by treatment of proteins with DNFB in 005 M Na2CO3 yielding DNP9-BGG, DNP55-MSH and DNP1o-OA.

Estimation of affinity by Stupp-Farr assay Affinities of the clonal antibodies were measured as described by Stupp, Yoshida & Paul (1969) and Willcox & McMichael (1975a). Aliquots of serum diluted in normal mouse serum diluted 10-fold with saline were titred as below for binding of Dl251P-DNP-lys. Dilutions giving about 70% hapten binding were then assayed at about six concentrations of hapten (usually between 10-6 and 2 x 10 - 9 M). To determine the concentration of antibody sites, the Scatchard plot was extrapolated to maximal hapten binding. The occupancy of the sites was then calculated and the Kd obtained from a Sips plot.

Isoelectric (pI) spectra of antibody to DNP Isoelectric focusing was carried out on thin layers of 5 % polyacrylamide gel with Ampholine carrier ampholytes (pH 5-8). Anti-DNP molecules were localized by coating the gel with 10 -8 M 13 1I-labelled a, N-(3,5-diiodo-4-hydroxy phenacetyl-N-(2,4-dinitrophenyl)-lysine (D 113 IP-DNP-lys). The method for fixing antibody-hapten in the gel, removal of excess hapten and autoradiography of dried plates has been previously described (Williamson, 1971). Thirty microlitres of serum samples were analysed, using pieces (6 x 15 mm) of Whatman 3-mm filterpaper for sample application. The product of a single

543

clone gave a characteristic family of protein bands (Askonas & Williamson, 1972b). Antigen-binding capacity (ABC) of serum The anti-DNP content of serum was determined by a modified Farr assay as previously described (Askonas & Williamson, 1972b). Serum samples at various dilutions (1/6, 1/36, 1/216, or for higher titre sera 1/11, 1/121, 1/1331) were added to 10-8 M D125IP-DNP-lysine and hapten bound by antibody precipitated with 50% saturated ammonium sulphate buffered to pH 7.4 with phosphate buffer. For each monoclonal antibody, standard curves of ABC at two-fold dilutions determined the slope used for calculation of the titres. Hapten binding was proportional to antibody concentration up to 15-20% hapten bound and then followed a log plot. ABC (antigen-binding capacity) was expressed as mmoles hapten bound per millilitre of serum at a hapten concentration of 1 x 10- 8 M. We present the geometric mean of individual titres, and negative sera are taken to have an ABC of 0 1. On the basis of two precipitation curves, an ABC of 2 was roughly equivalent to 5-10 pg anti-DNP which had an affinity of 10-7 mmoles/l. RESULTS Clonal dominance Clonal dominance is clearly illustrated by comparing the pI spectra of antibody to DNP in sera of mice after transfer of clonal spleen cells plus carrierprimed cells, or carrier-primed cells alone (Fig. 1). In the clonal transfers the clonal antibody product dominates the anti-DNP response almost completely, even after several boost injections over a 3-month period. Carrier-primed cells and DNP on the same carrier yield detectable anti-DNP only after a further injection of the same antigen in saline, but then the anti-DNP response in recipient mice is highly heterogeneous-showing the presence of polyclonal precursors cells for anti-DNP in the carrier-primed spleen cell population. Development of these clones has obviously been suppressed in the presence of the clonal cells. Our succeeding experiments are aimed at analysing the mechanism of this suppression. Properties of monoclonal antibodies to DNP available are given in Table 1. Only clones S13, S16 and Q10 were still available for transfer. First we investigated the transfer of two clones

Brigitte A. Askonas, A. J. McMichael & M. Estella Roux

544 pH 7.4

|[Clonal S13 S16

pH

b

5.5

pSample

1 2 3 45

1 2 3 4 5 6 7 Figure 1. pI spectra of antibodies to DNP after transfer of carrier-primed cells alone or with clonal spleen cells. Isoelectric focusing was carried out using pH 5-8 Ampholine. The autoradiographs show the uptake of D13 IP-DNPlysine by antibody to DNP in various serum samples (30 ul each). (a) 3 x 106 BGG-primed cells were transferred into irradiated mice with 10 pg DNP-BGG, the mice were boosted with I pg DNP-BGG 3 weeks later and serum analysed 10 days after the boost. 1-5: serum from five recipients. (b) 5 x 106 Spleen cells from mice carrying clone E9 transferred with 3 X 106 BGG-primed cells and 10 pug DNP-BGG serum 10 days after a boost injection of 10 pug DNP-BGG on day 27. 1 and 4-7: sera from five recipients; 2 and 3: sera from recipient 1, after a second and a third boost injection covering a period of 60 days.

differing in affinity to see whether one of the clones would dominate the response (Fig. 2). S13 and S16 differing in affinity by one order of magnitude were mixed in different proportions and transferred with Table 1. Properties of clones and their antibody to DNP Clone

Clone induced with:

IgG class

S13 S16 S24 Q10 E21

DNP-OA DNP-OA DNP-OA DNP-BGG DNP-OA DNP-BGG

I I I 1 1 I

E9

Kd* of anti-DNP (mmoles/1) 3 x10-9 2x10-8 1 x10-9

1-5 x 1O-7 3 x10-7 I

x10-7

Estimation of S clones and E21 were kindly carried out by Dr H. N. Willcox. * Kd assayed by Stupp-Farr assay, using D125-IP-DNPlysine (see the Materials and Methods section).

Figure 2. Transfer of mixed clonal populations. Spleen cells from mice bearing clones S13 and S16 were mixed in varying cell numbers as indicated on the figure (1-3-7-5 x 106). The cells and 50 pg DNP-OA were transferred into irradiated mice (eight to ten mice per group) and 10 days later the pI spectra of anti-DNP in the serum were analysed by isoelectric focusing. Number in each column indicate the number of sera positive for clonal antibody.

10 pg DNP-OA in saline. Ten days later, anti-DNP was characterized in serum by isoelectric focusing; pI spectra of antibody typical of both clones were present in all the sera of mice which had received mixed cell populations. Since we found previously (Askonas et al., 1972) that plaque-forming cells (PFC) arise in spleen only 6 days after transfer of the cells, it is reasonable that both clones can develop independently in the absence of antibody and proliferate and mature into immunoglobulin-secreting cells. Effect of autologous antibody on clonal antibody production To avoid the uncertainties of circulating antibodyantigen complexes and assure contact between clonal cells and antigen, the spleen cells were permitted contact with antigen in vitro before transfer into recipients treated with passive antibody. Spleen cells from mice carrying clone S13 and from ovalbumin-primed mice were incubated with DNP-OA for 60 min in vitro. Free antigen was then removed by centrifugation and the cells resuspended in medium were injected into irradiated syngeneic mice. Nine to 12 days later the serum was assayed for anti-DNP antibody by isoelectric focusing as well as by the Farr assay. 01 ml of S13 antibody administered i.p. 1 h before the cell transfer suppressed the triggering of clone S13; furthermore, when the recipients were boosted the suppression persisted although it was not permanent (Table 2).

Clonal memory cells Table 2. Suppression of S13 clonal antibody production by S13 antibody

Group DNP OA in vitro*

1 2 3

1-7

S13t Anti-DNP titre (ABC) (G.M.) anti-DNP Day 23 Day 42

-

+

-

+

Day0

0-1 25 0-4

1-6 9-6 4-5

x IO'

spleen cells (clone S13, transfer generation 5) and i.v. into 660 r-irradiated CBA/H mice (six mice per group). Mice were bled after 11 days, and the serum contained passive S13 antibody. The mice were boosted with 20 pg DNP-OA on day 14, and day 31, and bled after each boost on days 23 and 42. * Spleen cells incubated 60 min at 200 with 50 ,g/ml DNPOA and antigen removed by centrifugation before cell transfer into mice. t 0-1 ml S13 anti-DNP (ABC = 15/ml serum) administered i.p. 1 h before cell transfer. 3-9

x

106 OA carrier-primed spleen cells transferred

Q10 anti-DNP similarly suppressed clone Q10 transferred with BGG-primed spleen cells and DNP-BGG (not shown). This result demonstrated that suppression of the anti-hapten response could be achieved under these conditions with low levels of autologous antibody, and by antibody presumably identical to the receptor molecules. The possibility remained that suppression might in part be caused by antibody to the carrier protein or by co-existing anti-idiotypic antibody present in the sera. We therefore tested in later experiments suppression by heterologous monoclonal antibody, i.e. antibody to DNP produced by another cell clone which had been induced with DNP on a different carrier protein. Passive administration of antibody produced by other clones S13 clonal spleen cells were incubated with antigen, washed and transferred into irradiated recipients, pretreated with S24 or E21 antibody (0-1 ml ABC 20 x 10- 8 mM/ml). Assay as previously was by Farr test and IEF at 12 days, and again 10 days after a boost with DNP-OA. The results (Table 3) show a clear suppression by S24 antibody but not by E21 antibody. S24 possesses an affinity several fold higher than clone S13 whereas E21 antibody has a much lower affinity (two orders of magnitude). Thus suppression is not achieved by low levels of low affinity

545

Table 3. Effect of anti-DNP of lower or higher affinity on clonal development Day 0 antibody

No. of mice

Serum ABC* (G.M.) S13 antibody

None E21 S24

6 6 6

1P67 2-7 04

Spleen cells (7 x 106) from mice carrying S13 clone and 3 x 106 OA-primed spleen cells were transferred with 10 pg DNP-OA into irradiated mice. * Serum titre on day 47 after cell transfer. Recipient mice were boosted with DNP-OA on day 36.

antibody, but higher affinity antibody is very effective. To ascertain that suppression is effected by the monoclonal anti-DNP and not by antibody directed to the carrier protein, we also tested antibody from clones induced and triggered with DNP-OA (whose serum might also contain some antibody to OA) on clone Q1O induced with DNP-BGG. This combination yielded a suppression persisting for two boost injections but as previously noted, after antigen excess clonal antibody production was triggered, and the antibody suppression thus was reversible (Fig. 3). Clonal pI spectra are visualized in Fig. 4. After the first boost, four out of five control mice had strong Q1O antibody, whereas only two out of five of the antibody-treated mice had trace amounts of Q10 antibody. On recovery four out of five mice in both groups produced Q1O antibody. The IEF results also showed that S13 antibody persisted at the time of the first bleed and its characteristic spectrotype could be seen, confirming that conditions were of antibody excess over antigen. Time of administration of passive antibody To test the effect of antibody during various stages of clonal expansion and maturation, passive antibody was given at different times after triggering the transferred clone. Experimental conditions were the same as those described above, except that passive antibody was given either 1 h before cell transfer, or 4 days after. S13 antibody suppressed clone Q10 when given on day 0. When given on day 4, enhancement of the antibody response was found (Figs 3 and 4). The same antibody could therefore either suppress or enhance depending on the timing

Brigitte A. Askonas, A. J. McMichael & M. Estella Roux

546 80r A

60 E An

IQ10 E

co

401-

10

; Passive J S13 30

0

aII

0-4*-

0

10

30 40 50 Boost Boost Days after transfer Figure 3. Effect of S13 antibody on the production of antibody by clone Q10. Irradiated mice (six per group) received i.v. 7-5 X 106 spleen cells (clone Q10 DNP-BGG-induced) and 2-2 x 106 BGG-primed spleen cells after incubation of the cell mixture with antigen in vitro. Antigen in the supernatant was removed before cell transfer by centrifugation. Serum antibody titres (ABC) in: (0) control mice, no antibody; (X) 0-1 ml S13 antibody i.p. (ABC = 20mM x 10-8 hapten/ ml) 1 h before cell transfer; (A) 01 ml S13 antibody (as above) 4 days after cell transfer. ABC is expressed as geometric mean of titres of individual sera. Boost injections of 10 pg DNP-BGG were given at times indicated.

20

of administration. We have since tested day 4 antibody in other clonal transfers and this enhancement is rather variable, but suppression was never found. Antibody given on day 2 had little measurable effect. The effect of passive antibody

on

immunological

memory

Triggering of B cells by antigen results in simultaneous generation of PFC and memory cells (Askonas & Williamson, 1972b), so that quantification of antibody production is only a measure of a part of the immune response. Retransfer of clonal B cells can be used to estimate memory, either by performing a limiting dilution analysis or measuring the amount of antibody produced after a second transfer. Q10 memory cells were transferred under identical conditions to those above, and its clonal antibody was given on day 0 or day 4; on day 28 three mice from each group served as spleen cell donors for retransfer with antigen. The several sets of recipients were bled 14 days later and assayed for Q10 antibody. The results shown in Table 4 indicate that memory was suppressed in parallel with PFC generation, when antibody was given on day 0. Day

|-Sample

5 +

5

DNP-BGG+

6

3

+

vitro S13 antiDay Day DNP-OA 0 4 Q10 cells + BGG-primed cells + DNP-BGG in vitro. Spin. Transfer. Figure 4. Analysis by isoelectric focusing of clonal expression after antibody treatment. Conditions of transfer of clone Q1O, incubation with DNP-BGG and treatment of mice with S13 antibody as described in Fig. 3. Autoradiograph of isoelectric focusing analysis using pH 5-9 ampholyte carrier, gel overlaid with radioactive 1311-labelled DNP-hapten (see the Materials and Methods section).

in

4 antibody in this case did not enhance. This result suggests that the suppression by antibody affects cells at the memory cell level. Table 4. Effect of antibody on memory generation

First recipients Q anti-DNP Day0 Day 4

ABC* 29 5 5-6 23 5

Second cell transfer Serum titre IEF-positive Q pI spectrum ABC* 10 0 9/10 50 5/10 11 0 7/9

First recipients received 8-5 x 106 Q spleen cells+4 x 106 BGG-primed spleen cells, after incubation with 50 pg/ml DNP-BGG in vitro; passive Q1O antibody (ABC = 3 x 10-8 mM hapten) given as indicated and serum titred 11 days later. Second transfer of spleen cells (2 x 107) from above recipients (three from each group) into ten irradiated mice with 10 jug DNP-BGG 28 days after original cell transfer. (ABC after 10 days.) * ABC was the geometric mean of titres expressed as hapten bound (mM) x 10- 8/ml serum.

547

Clonal memory cells (1'4) E

10

0

}Q1pO

05 b-

0

E E

A

B

C

Figure 5. Antigen-treated T or B cells and the effect of antibody. 7 x 106 Spleen cells from Q10 donors (B cells) and 3 x 106 MSH-primed cells (T cells) were transferred i.v. into irradiated mice. The groups varied in regard to the cell types incubated with antigen in vitro (see below). ABC represents geometric mean of anti-DNP titres in mm x 10-8 hapten/ml serum on day 30, 10 days after i.p. boost with 1 pg DNP-MSH. Open columns, control cell transfers; hatched columns, mice received S24 anti-DNP (ABC = 1-5) i.p. 1 h before cell transfer. (A) Q10 cells and MSH cells incubated in vitro for 1 h at 200 with 5 pg DNPMSH. Antigen was removed by centrifugation. (B) Only the anti-hapten B cells (Q10) were incubated with DNP-MSH. Free antigen was removed and fresh T cells admixed before transfer. (C) Only MSH-primed cells were incubated with DNP-MSH, antigen was removed, and Q10 cells were admixed.

Separate incubation of T and B cells with antigen Anti-hapten production in this system is entirely T dependent (Askonas & Williamson, 1972b) and to resolve which cell type (B or T cells) is susceptible to antibody suppression after antigen uptake, we used DNP on a different carrier protein (Maia Squinado haemocyamin (MSH)) as antigen, and restored the anti-DNP response with spleen cells from mice primed with MSH. This enabled us to expose one cell type, either B cells (anti-DNP) or T cells (antiMSH) to antigen (DNP- MSH), prior to transfer of both co-operating cell types. Fig. 5 illustrates such an experiment: antibody passively administered was derived from clone S24 in this instance and possessed a very high affinity. (Kd = 1 x 10-9 mmoles/l). A clear requirement for MSH-primed cells when the antigen was changed to DNP-MSH was demonstrated, as expected (not illustrated). We obtained the usual suppression of Q10 clonal antibody production when both T and B cells had been pre-incubated with antigen before transfer into an irradiated host treated with passive antibody. However, B cells after

| Sample

S13 antiDNP-OA Day 0 Q10 cells + DNP-MSH in vitro. Spin. Add MSH-primed cells. Transfer Figure 6. Antigen-free T cells lead to enhancement of clonal antibody production after passive antibody. Autoradiograph of isoelectric focusing of serum anti-DNP from two groups of the experiment described in Fig. 5. Spleen cells of Q1O donors were incubated with DNP-MSH for 1 h at 200 and antigen removed by centrifugation. Fresh MSH-primed spleen cells (3 x 106) were admixed prior to transfer of 7 x 106 QIO cells into irradiated mice. The second group of recipients received S24 antibody (ABC = 1-5) 1 h before cell transfer.

antigen uptake and transfer with fresh T cells into antibody-treated hosts were triggered into production of Q10 antibody. Presumably, the T cells were free to interact with the determinants on the carrier protein associated with the B cells and cell-cell co-operation thus could occur (Fig. 7). If anything, antibody caused an enhancement of anti-DNP production compared to the control group (Fig. 5). This is visualized by the autoradiograph of the pI spectra of individual mouse sera from the two groups (Fig. 6). Treatment of T cells alone with antigen did not block B-cell reactivity in the presence of antibody, when the B cells had not taken up antigen

(Fig. 5). DISCUSSION The use of cloned anti-DNP memory cells and mono-

clonal antibody have enabled us to demonstrate

Brigitte A. Askonas, A. J. McMichael & M. Estella Roux

548 B

and T cells + antigen, then antibody

No antibody B cells+ antigenthen T cells and antibody

B cell

T cell

Ant ibody

Figure 7. A model for antigen-antibody mediated blockade. () Anti-DNP; (A) DNP-hapten group; (stippled areas) carrier determinant recognized by T cells. After antigen uptake by both T and B cells, anti-DNP blocks antibody production and T- and B-cell cooperation. After Ag treatment of the B cells only, anti-hapten does not block T-cell recognition and antibody induction.

several new aspects of antibody-mediated control of the immune response. The experimental design has ensured contact between cells and antigen and conditions of antibody excess, even though antibody was passively injected at low (,g) levels. Demonstration of the passive anti-DNP in serum by IEF 10 days later has confirmed this. This model seems appropriate since in vivo cell receptors have no difficulty in taking up antigen in the presence of antibody (Bystryn, Siskind & Uhr, 1975). In fact antibody enhances this uptake. The experiments show clear suppression of S13 clonal anti-DNP production, corresponding to secondary responses, by the clones' own antibody. This suggests that competition for antigen between receptor S13 antibody and free S13 antibody cannot account on its own for the suppressive effects. This and the fact that only cell-bound antigen is exposed

to antibody indicates that some sort of complex is being formed on the cell surface. Persistence of the suppression for several days, often through the first boost injection suggests either that the complex persists for some time on the cell surface without interiorization and blockades cell receptors as proposed by Diener & Feldmann (1970) for tolerance induction; or alternatively, that this complexreceptor interaction affects cellular events for a finite period of time. The blockade is reversible ultimately with further antigen administration, clonal antibody with its characteristic pI spectrum is produced and we are therefore not dealing with clonal deletion. The chance of recruiting a new anti-DNP clone with the same antibody phenotype in several experiments is minute, in view of the large repertoire of antibodies to hapten in CBA/H mice (Kreth & Williamson, 1973; Pink & Askonas, 1974). Higher affinity antibody is also effective, in line with suppression experiments in vivo (Werblin & Siskin, 1972). By changing the carrier protein and the source of clonal antibody we can draw two further conclusions. First, the experiments exclude any role for antibody to carrier protein in the suppression. Secondly, we cannot assign any role to anti-idiotypic antibody in these experiments. This sort of regulatory mechanism has been proposed (Rowley, Fitch, Stuart, Kohler & Cosenza, 1973; Jerne, 1974) and heterologous anti-idiotype has been shown to suppress idiotype expression (Hart, Wang, Pawlak & Nisonoff, 1972; Cosenza & Kohler, 1972; Eichmann, 1974). However, shared idiotypes between S13, S24 and Q10 are highly unlikely and many attempts to deliberately induce idiotypic antibodies to our clonal products so far have proved unsuccessful. In our case IgGI antibody to hapten suppresses although it does not fix complement. This contrasts the observations by Eichmann (1974), that only

complement-fixing IgG2 guinea-pig anti-idiotypic antibody is suppressive. On the other hand, the mechanism of antibody inhibition is different in the two cases, and in our experiments is probably effected by preventing T- and B-cell co-operation (see below). We have no data concerning possible T suppressor cells (Gershon, 1974; Eichmann, 1975) in our system. Inhibition of the immediate secondary response is mediated by antibody, but we are unable to exclude that antigen-antibody on cell surfaces may be efficiently inducing transient suppressor cells to account for the extended period of suppression. Suppression by lower affinity antibody, E21, given

Clonal memory cells in the same microgram amount as S24, was not achieved. In the same experiment S24 suppressed. Limitation in availability of monoclonal antibody did not allow us further to study this aspect. Passive antibody also decreased memory generation in parallel to PFC induction (Table 4) indicating that antigen-antibody blockade also acts on initial triggering of B memory cells. The reversibility of antibody suppression may well be responsible for the preservation of a pool of memory cells even on continued antigenic stimulation. Exhaustion of clonal memory cells into mature short-lived immunoglobulin-secreting cells would be deleterious to the defence system; though clonal memory cells may be temporarily suppressed, they are long lived cells and thus will be preserved in contrast to early immature DNP-specific B cells which could easily be deleted by the antigen-antibody blockade since they are short-lived (Strober & Dilley, 1973) and appear unable to regenerate immunoglobulin receptors after capping (Sidman & Unanue, 1975; Raff, Owen, Cooper, Lawton, Magson & Gathings, 1975). Thus once established, a clone may dominate the antibody response by inhibiting priming of new precursor B cells. Enhancement of the transferred clonal antibody response by late administration of passive antibody was an unexpected finding. We are unable to explain this adequately, and it has not been observed in all of our experiments. It is possible that antibody given on day 4 affects T suppressor cells more than T helper cells, already activated by day 4. Alternatively, the antigen requirement of the B cell may be different on day 4 compared to day 0. The latter explanation receives support from experiments with DNP-pneumococcal polysaccharide which dramatically suppresses the appearance of PFC when given on day 0 with antigen, but does not affect the antiDNP response when given 4-5 days after cell transfer (Mitchell et al., 1972) and has less effect on memory generation than antibody formation (Klaus & Willcox, 1975). An easy explanation would be that T-B cell co-operation is only important during the first few days but other data are not in line with this view. Thymus replacing factor appears to act on maturation of B cells (Askonas, Schimpl & Wecker, 1974; Dutton, 1974) and a thymus dependent response is restored when activated carrier primed cells are administered on day 4 (Askonas & Roux, unpublished results). It is however not unreasonable that antibody does not suppress activated clones as soon

549

as it is produced and that feedback inhibition of B cells only operates at the earlier triggering phase. Enhancement was also found when conditions were arranged such that T cells were not exposed to the antigen in the incubation in vitro. This finding gives some insight into the mechanism of antigenantibody blockade. A possible model is outlined in Fig. 7 and is as follows: under conditions where both B and T cells are incubated with the appropriate hapten-carrier, antigen is taken up by both cell types and transferred into irradiated recipients. On subsequent exposure to anti-DNP antibody, receptors on both B and T cells are blocked by antigen-antibody and cell-cell co-operation is prevented, and suppression of antibody produced is always observed. On the other hand, when the T cells are not exposed to antigen prior to cell transfer, B cells presumably coated by antigens plus antibody are triggered. Our model suggests that under these latter circumstances, the T cells can still recognize carrier determinants on the B cell-bound antigen and B-cell-T-cell co-operation can complete the B-cell triggering. The enhancement in these responses (Figs 5 and 6) indicates that anti-hapten, if anything favours the firm binding of antigen to the B cells and more efficient T-cell triggering ensues. This model makes the a priori assumption that T cells recognize and bind antigen directly. There is increasing evidence that this is true, although whether T-cell recognition sites share determinants with immunoglobulin molecules (see Eichmann & Rajewsky, 1975), or are a product of the I region of the major histocompatibility complex (see Benacerraf & McDevitt, 1972) is still unresolved. Kontiainen & Andersson (1975) have specifically enriched antigen specific helper T cells using a rosetting technique; furthermore, helper T cells can be specifically eliminated by radioactive antigen suicide (Roelants & Askonas, 1971; Basten, Miller, Warner & Pye, 1971), although recently Basten, Miller & Abraham (1975) have encountered difficulties in achieving antigen suicide in a pure T-cell population. In conclusion, therefore, our results show clearly that homologous and higher affinity anti-hapten antibody suppresses antihapten clones. Anticarrier and anti idiotype effects are excluded. The antibody acts on B-memory cells, and B precursor cells, and T cells after antigen binding and probably suppresses by inhibiting T- and B-cell co-operation. The effect of antigen-antibody is temporary and reversible, and thus favours the preservation of a memory cell pool

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rather than priming of new clones developing from early, short lived precursor cells. The timing of antibody administration indicates that clones already triggered in the first 4 days after antigen contact cannot be suppressed by antibody.

ACKNOWLEDGMENTS We thank Jackie Welstead and Dianne Millican for their excellent assistance.

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