Idiotype-specific regulation might contribute to specific unresponsiveness in dextran-primed mice

(~) ELSEVIER Paris 1989

Res. Immunol. i989, i40, 7-i8

IDIOTYPE-SPECIFIC REGULATION MIGHT CONTRIBUTE TO SPECIFIC UNRESPONSIVENESS IN DEXTIL~N-PRIMED MICE I. Lundkvist (l)

(2),

~)o Portnoi

(2)

and A. Coufinho

(2)

(1) The Unit for Applied Cell and Molecular Biology, Umed University, S-901 87 Limed (Sweden), and

(2) Laboratory o f lmmunobiology, lnstitut Pasteur, 75724 Paris Cedex 15

SUMMARY

Priming of adult responder mice with optimal immunogenic doses of dextran ~,1-6 results in reduced antibody responses to secondary antigenic challenge. We have now quantitated dextran-specific clonal precursors in the large and small B-lymphocyte compartments within several days or several months after priming with either dextran or antiidiotypic antibodies directed to a dominant idiotype of C57BL/6 mice which accounts for more than 50 °70 of the antibody response. The results show that ~ secondary unresponsiveness >> correlates with idiotype-directed depletion of the appropriate specificities from the immunocompetent resting B-cell pool. KEY-WORDS"

Idiotype, Dextran B512, B lymphocyte; hnmunoregu!ation,

Depletion.

INTRODUCTION We recently analysed the available and actual antibody repertoires of C57BL/6 and BALB/c mice in terms of both paratopic anti-dextran B512 (Dex) specificities and B cell clonal precursors carrying an idiotype (Id) dominating specific antibody responses in the C57BL/6 strain, but which is produced as a ~ natural >>antibody in BALB/c mice [1, 2]. By direct quan-

Received January 3, 1989. Correspondance to: Inger Lundkvist, Department of Immunohematology and Bloodbank, University Hospital, Building 1, E3-Q, Rijnsburgerweg 10, 2333 AA Leiden (The Netherlands).

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L L U N D K V I S T A N D COLL.

titation of clonal precursors among resting or ~ naturally)) activated B cells, we found evidence for idiotypic regulation in the determination of which clones are selected into an antigen-specific immune response from the pool of available specificities [3l. With this information in mind, we have now addressed the question of reduced specific antibody responses obtained after secondary challenge with antigen. This phenomenon was previously described by Fernandez and M611er in Dex responses and they suggested that it results from the production of autoantiidiotypic antibodies in immune mice [4]. A variety of experiments on the organization and regulation of available repertoires have, however, suggested the importance of systemic rather than clonal properties in controlling the fate of activated cells [5]. We have previously shown that, depending upon the state of activation of the specific precursor cells, the kinetics and magnitude of a primary response differ considerably [3]. Thus, large activated cells are not easily induced to produce an immune response, and we have suggested that regulatory network-like interactions - - operating in the pool of activated cells and involving both paratopic and idiotypic specificities - hamper the laxge clonal amplifications required for an effective response. Priming of C57BL/6 mice with Dex results in blast transformation of small resting precursors and subsequent production of high levels of anti-Dex antibodies, the majority of which carry the 17-9 Id [6]. We report here that Dexpriming has long-lasting consequences on the absolute numbers of Dex-specific precursors such that, six weeks after priming, the number of precursors has decreased five-fold compared to normal, non-immunized mice. This depletion concerns mainly the pool of small resting cells and appears to be due to !d-specific regulatory mechanisms.

MATERIALS AND METHODS Mice.

C57BL/6 mice 8-10 weeks old and Fisher strain rats were obtained from the animal facilities at the Institut Pasteur. Antigens, antibodies and immunizations.

Native Dex B512 was given i.p. at a dose of 10 l~g per mouse. Lipopolysaccharide (LPS) was obtained from Salmonella abortus-equi (Difco Laboratories Inc., Detroit, MI). The production a~d characterization of the 17-9 Id antibody (anti-

Dex ELISA HA Id i.p.

= = = = =

d e x t r a n B512. enzyme-linked immtmoadsorbent assay. haemagglutination. idiotype. intraperitoneal(ly).

LDA LPS PFC RIA SRC

= limiting d i l u t i o n analysis. = lipopolysaccharide. = p l a q u e - f o r m i n g cell. = radioimmunoassay. = sheep e r y t h r o c y t e .

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dextran ~,1-6; 0t,k) and the VH3.G12.E6 anti-Id antibody (anti-17-9; "r~,k) have previously been described in detail [1]. The anti-Id antibodies were co-poiymerized with the thymus-independent antigen LPS using glutaraldehyde as previously described [2] and immunized i.p. at a dose of 5 ~g per mouse. As control, a separate group of mice were immunized with LPS alone, treated in the same way as conjugated LPS.

Preparation and fractionation of spleen cells. Murine spleen cells were prepared as previously described [7] and fractionated cn discontinuous Percoll gradients [6, 8]; cell fractions with densities 1.060 ~ p < 1.070 are referred to as large cells, while those with p > 1.070 are referred to as small cells. In every experiment, the purity of small and large cell fractions was controlled in a cell-size analyser (Coulter Counter 2M, Coultronics, Margency, France).

Limiting dilution analysis (LDA). The preparation of growth-supporting rat thymocytes as well as cell culture methods for LDA have been described previously [7]. Briefly, limited numbers of unseparated, small, or large spleen cells, respectively, were seeded in 0.2 ml cultures containing 3 x 105 rat thymocytes in RPMI medium supplemented with 5 × 10 -5 M 2-mercaptoethanol, glutamine, antibiotics, 10 °7o foetal calf serum (Gibco Biocult, Glasgow, Scotland) and LPS at 25 ~g/ml. Six different cell concentrations, each represented by 48 identical cultures, were set up for each frequency determination, and another 48 cultures containing rat thymocytes and LPS but no spleen cells were used as negative standard in all experiments.

Enzyme-linked immunosorbent assay (ELISA). The presence of total IgM and Dex-specific IgM antibodies in culture supernatants and serum were determined, as previously described in detail [9], by .coating the wells of microtitreplates (Nunc, Roskilde, Denmark) with purified goat anti-mouse Ig (Cappel, Cooper BioMedical Inc., Malvern, PA) or DexT-2000 at 10 ~tg/ml (Pharmacia, Uppsala, Sweden). After saturation with 1 07o gelatin in phosphate-buffered saline (PBS), the supernatant/serum was added and bound antibodies were revealed by the addition of peroxidase-labelled goat anti-mouse IgM antibodies (Southern Biotechnology Inc., Birmingham, AL.). The Dex-titre in serum was determined by comparing the absorbance of the sample with that of a standard curve obtained by adding known concentrations of the anti-Dex monoclonal antibody D. 16 (~,k) in the same assay.

Haemagglutination (HA). The anti-Dex titre in mouse serum was alternatively determined in HA using Dexcoated SRC mixed with serial two-step dilutions of the different sera. The highest dilution at which agglutination could be observed was considered as the anti-Dex titre. In parallel, the anti-SRC titre was recorded using non-coated SRC and subtracted from the figures reported for anti-Dex titres.

Radioimmunoassay (RIA). The presence of 17-9 Id-positive antibodies in culture supernatants and serum was determined in a pr~:iously described competitive RIA [1] using 125I-labelled

10

I. L U N D K V I S T A N D COLL.

VH3.GI2.E6 anti-Id antibodies [10]. The amount of 17-9 Id equivalents in serum was determined by comparing the serum dilutions to known concentrations of Id giving the same number of cpm.

RESULTS

Secondary unresponsiveness to Dex after priming with either Dex or anti-ld antibodies. Anti-Dex antibodies are easily induced in the primary response of C57BL/6 mice to either Dex or anti-Id antibodies to the dominant anti-Dex Id (i 7-9). We have previously shown that immunization with 5 l~g of a conjugate of VI-I3.GI2.E6 anti-Id antibodies and LPS specifically activates 17-9 Id-positive B lymphocytes, irrespective of their paratopic specificity [2, 3]. Since roughly 50 070 of all 17-9 Id-positive precursor cells in C57BL/6 mice are specific for Dex, anti-Id-LPS priming results in high titres of anti-Dex antibodies in the serum of immunized mice [2]. We found it interesting to investigate whether such anti-Dex responses induced in the absence of Dex also give rise to reduced secondary responses. Thus, one of the mechanisms invoked to explain secondary unresponsiveness to polysaccharides is based on the persistence of the antigen. C57BL/6 mice were primed either with Dex or the anti-Id-LPS conjugate. Roughly two months later, the mice were bled and subsequently immunized with Dex. Figure 1 shows the serum titres of anti-Dex antibodies before (day - 1) and after (day 1-13) the Dex boost. Compared to the titres observed in a vrimarv anti-Dex resnonse (fi~_ 1A), nrimin~ with alth~r 1 3 ~ Y n r t h p anti-Id-LPS conjugate results in strongly reduced anti-Dex responses, reaching levels which are only 22 and 30 °70,respectively, of the primary response. The extent of unresponsiveness to Dex after anti-Id priming is surprising. Thus, considering that only half of all Id-positive B-cell precursors are actually antiDex [2], together with the fact that priming with Dex does not result in the complete absence of a secondary response, it would be expected that at least 50 °70 of the Dex-reactive clones would be available to produce a secondary response. As shown in figure 1C, this is not what we find, and the results suggest that Id-directed manipulations trigger regulatory mechanisms which modulate Id-negative Dex-spec~fic precursors as well. --

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Frequencies of B-cell clonai precursors producing Dex-specific antibodies among large and small spleen cells in normal and primed mice. Previous studies in this system have shown that thero is a correlation between the state of activity of the antigen-specific/Id-positive precursors and their contribution to a conventional immune response. While the large, ~ naturally)) activated l_~vnphoc~es are involved in internal and self-determ_ined

MECHANISMS FOR B-L YMPHOCYTE TOLERANCE

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FIG. I. ~- Serum titres o f anti-Dex antibodies before and after Dex boost. Groups of C 5 7 B L / 6 mice were immunized with (A) NaCl, (B) Dex or (C) anti-Id-LPS conjugate; 8-9 weeks later, all mice were immunized with Dex. The mice were bled one day before and at several timepoints after the second immunization. The anti-Dex titres were calculated as described in (>. Open circles represent individual mice and ~.,.,a~. ..!..-_-! ~_:-_,_. . . ~ show mean values.

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L L U N D K V I S T A N D COLL.

activities, the small resting immunocompetent cells - - which comprise 85-90 °70 of the total SC population - - primarily account for specific antibody responses, as they are available for engaging in large clonal expansions and differentiation to high-rate antibody secretion upon stimulation with foreign antigens. Thus, Dex priming of C57BL/6 mice results in the production of Dex-reactive, 17-9 Id-positive antibodies. BALB/c mice, on the other hand, respond to Dex using a population of Dex-specific precursors that are Id-negative - - although they contain Dex-specific Id-positive B cells in spleen which, when appropriately triggered, produce this type of antibody [2]. This discrepancy was found to be correlated with the differential distribution of Id-positive precursors among small and large cells in the two strains" in C57BL/6 all Id-positive precursors are found in the small cell population, while the opposite situation is found in BALB/c [3]. Being aware of this relationship, we decided to analyse the state of activation of the Dex-specific precursor cells at the timepoint when the mice were given a second immunization. As shown in table I, when the absolute frequency of Dex-specific precursors was determined late after Dex- and antiId-LPS-priming, there was a marked decrease in the number of specific clonal precursors among SC compared to non-immunized mice. The clear-cut decrease in the absolute number of specific precursors in the small cell pool was not, however, completely accounted for by the increase observed among the large cells. The titre of Dex-specific antibodies found in serum after a secondary challenge with Dex (fig. 1B) was thus in accordance with the total number of Dex-reactive precursors present in spleen at that time. Furthermore, the finding that priming with anti-Id antibodies reduced the absolute frequency of Dex-specific precursors among total SC by roughly 50 °70, ~,-._,~,~r,4o

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precursors in C57BL/6 mice are 17-9 Id-positive [2]. The severe depletion observed in the small cell pool suggests, however, that not only 17-9 Id-positive anti-Dex clones, but also those carrying other Id, were influenced by this treatment.

TABLE I. - - Absolute frequencies of Dex-specific precursor B cells in total, small and large spleen cells from normal and primed C57BL/6 mice.

Normal mice 6 weeks after Dex priming 12 weeks after anti-Id-LPS priming The ,J---v ~ C ~

Small

Large

Total

11900 1/4,000 1/3,670

1/ 1,550 1/2,300 1/ 1,400

1/ 1,200 1/5,000 1/2,860

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M E C H A N I S M S FOR B - L Y M P H O C Y T E TOLERANCE

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Analysis of available and actual paratopic and idiotypic repertoires in Dex and anti-Id-LPS primed mice. In the next set of experiments, we took advantage of the fact that Dex specificity and 17-9 Id expression are closely associated parameters in C57BL/6 mice. Criss-cross-type experiments, where the effects of Dex-priming on Id expression - - and vice versa - - are performed early after priming provide possibilities to evaluate the immediate consequences of priming, and such information could be useful for the interpretation of events observed after secondary challenge. We therefore primed C57BL/6 mice with Dex or the anti-Id-LPS conjugate and determined the absolute frequencies of both Dexspecific and Id-positive precursors in spleen 5 days later. We previously demonstrated the in vivo functional significance of the results obtained in LDA [6] and here we extend this finding by showing that priming with antigens directed either to paratopic or idiotypic epitopes resulted in blast transformation and disappearance from the small cell pool of the precursors carrying complementary structures (table II). As expected, Dex priming resulted in activation of many Id-positive precursors, as shown by the decrease among small cells and the increase among large cells of the absolute frequency of Id-positive precursor cells. As also shown in table II, priming with anti-Id antibodies activated a good half of the small, resting Dex-specific precursors to become large, which is in line with the previously established correlation between Dex-specificity and 17-9 Id expression in this strain. In conclusion, these experiments show that priming with either of these two thymusindependent antigens resulted in profound depletion of the appropriate cional specificities from the resting B-cell pool, even early after immunization in

TABLE II. - - Absolute frequencies of Dex-specific and 17-9 Id-positive precursors in C57BL/6 mice 5 days after priming.

anti-Dex Normal mice

S L T Dex-primed mice S L T anti-ld-LPS l.,rimed mice S L T

1/500 1/1,550 1/1,200 < 1/10,000 1/1,259 1/2,700 1/1,450 1/1,620 1/1,400

Id-positive 1/750 <

1/10,000

1/780 1/2,300 1/4,000 1/2,950

< I/i0,000 1/2,500 ND

Groups of C57BL/6 mice were primed with Dex or the anti-ld-LPS conjugate or left un~t~ated; 5 days later, mice were sacrificed and spleen cells prepared and fractionated as described in ~Materials and Methods,. The absolute frequencies were determined and_ calculated as described in table I. ND = not determined.

14

I. LUNDK~,'.~T A N D COLL.

decreased total frequencies of specific precursor cells. Moreover, the ~ crisscross >>frequencies observed early after priming quite adequately reflected the paratopic and idiotypic composition of the available repertoires and the in vivo immune response in this strain. 17-9

Id expression

in s~ondary

anti-Dex

responses.

What then is the idiotypic profile of the anti-Dex antibodies produced in a t~secondary >) immune response? Figure 2A shows the kinetic profile of 1 7 - 9 Id serum titres after primary immunization with Dex. Figure 2B shows that the Id titres found in serum after secondary Dex immunization were only 15 % of the titres observed in the primary response. Although this low Id level was in general accordance with the low titres of anti-Dex antibodies (fig. 1B), the expression of the 17-9 Id appeared selectively tt suppressed>> in

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M E C H A N I S M S FOR B-L Y M P H O C Y T E T O L E R A N C E

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TABLEIII. -- Absolute frequencies of i%9 |d-positive precursor B cells in small and large spleen cells from normal and primed C57BL/6 mice.

Normal mice 6 weeks after Dex priming 12 weeks after anti-Id~LPS priming

In small cells

In large cells

1/750 1/5,000 1/10,000

< 1/10,000 1/2,280 i/1,400

The frequencies were determined in pools of spleen cells from 3-4 mice. The absolute frequencies were calculated by correcting the frequency of Id-positive precursors with the frequency of IgM-producing precursors in the same cell suspensions.

Dex-primed mice. These indications were directly supported by the results of clonal precursor frequencies shown in table III. Thus, very low numbers of Id-producing B cells were found in the small lymphocyte fraction after Dex-priming. If only antigen-specific mechanisms were involved in secondary unresponsiveness to Dex, it would be expected that about half of all Id-positive precursors would be unaffected, since they were not Dex-binding antibodies [2]. As can be seen, however, only 15 % of the Id-producing precursors were left in this compartment after Dex-priming, showing that Id-directed mechanisms operate in antigen-induced unresponsiveness. In previous reports, we have shown and discussed the effects of anti-IdLPS priming of C57BL/6 mice [2, 3]. Thus, as late as three months after priming, all Id-positive precursor cells were still found in the activated cell population and the serum of such mice contained low but detectable levels of 17-9 Id antibodies ([3] and fig. 2C). It was therefore not surprising to find ,,,,~, ,i. ,,,~. . o~1 ,t,~, . . . .uul . titres ., .~n,mn~u . . :--'~ practica lly unaltered even after Dex immunization of these mice, although there was a clearly detectable anti-Dex antibody response. As strongly suggested from these results such anti-Dex responses in anti-Id-primed mice were Id-negative. Thus, the binding of serum antibodies to Dex cannot be inhibited by anti-Id antibodies (data not shown), in contrast with anti-Dex antibodies obtained in the primary response of normal C57BL/6 mice.

DISCUSSION

The present experiments have attempted to analyse the cellular basis for specific secondary unresponsiveness to Dex in responder mice. This phenomenon, which has been previously described [4] and is confirmed here, is consistently observed in responses to thymus-independent polysaccharides [11, 12]. Its clinical significance is shown by the regularly increasing difficulty of repeatedly infected patients (e.g. with Gram-positive bacteria) to limit the course of the disease [13].

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L L U N D K V I S T A N D COLL.

Various mechanisms have been proposed to explain this phenomenon, such as suppressor T cells [14], autoantiidiotypi¢ antibodies [4, 15] and antigen persistence followed by high-zone tolerance of specific B cells [16]. Thus far, however, the cellular basis for unresponsiveness, as analysed at the level of immunocompetent B cells, had not been reported. Such an analysis could help in determining the site and nature of deficiency in primed individuals (e.g. elimination, blockade or suppression of B cells versus normal antigen-specific B-cell compartments modulated by regulatory mechanisms). Ideally, the study should consider both the paratopic and the idiotypic profiles of available B-cell repertoires in primed mice, since this could give indications as to the nature of the mechanisms determining unresponsiveness (antigen- versus idiotypedirected). We undertook this analysis in light of our previous observations of Dexrelated antibody responses in C57BL/6 mice. Our methods, based upon LDA of clonal precursors in vitro, adequately reflect in vivo conditions and measure the numbers of functionally antigen-reactive B cells [6]. Furthermore, we know that specific in vivo responses are primarily correlated with the number of antigen-specific B cells in the available resting lymphocyte compartment; large B cells with the same reactivities contribute comparatively less to those responses [3]. Finally, we had established that normal C57BL/6 mice contained high numbers of resting anti-Dex B cells in their spleen [6], half of which expressed the 17-9 Id [2] and that, conversely, only half of all Id-positive cells were actually anti-Dex. More importantly, we had observed good correlation between such clonal frequencies and both the idiotypic profile of primary anti-Dex responses and the paratopic repertoire of antibody responses triggered with anti-Id-LPS conjugates. Present experiments confirm some of those conclusions. Thus, in the various situations ~ n a l y s e d , o n n d e n r r e l a t i n n ~Yi~t~ h o t w p o n Fronllonrloc n f ~nti_rloY t~r

Id-positive precursors in the small cell pool and antigen-induced antibody or idiotypic responses measured in serum (e.g. anti-Id-LPS-primed ,,nice contain only about 5 % of the Id-positive precursors of control mice in the small cell pool, and the serum Id response after Dex immunization is also around 5 % that of normal mice). Furthermore, repertoire analysis shortly after priming (day 5) with either Dex or anti-Id reveals the depletion of corresponding reactivities from the smaU cell pool, with little influence upon the ~ parallel >>set of precursors. Thus, Dex priming depletes only 2/3 of Id-positive precursors, leaving a considerable number of these cells (which are not anti-Dex) in the small cell fraction. The converse is true in anti-Id-primed mice, where more than 1/3 of anti-Dex, Id-negative precursors remain in the available repertoire. Interestingly, however, this distinction between the two <~parallel>> sets leads to a decrease in both, and ~ cross-influences >>are clearly detected. Thus, several weeks after priming, only 15 % of the parallel set of precursors are left in the available repertoire in both cases, and there is not much difference in depletion between the precursor set selectively addressed by priming and the parallel set. In other words, Id-directed depletion has long-term effects which result in elimination of Dex-specific but id-negative cells, and vice versa. The reasons

M E C H A N I S M S F O R B-L Y M P H O C Y T E T O L E R A N C E

17

for these effects are not clear at present, but they may well be antigenindependent, since anti-Id-primed animals were not exposed to Dex before LDA were carried out. Furthermore, it is unlikely that the low dose of anti-Id antibodies injected with the conjugate would persist for 12 weeks in the animals. It is likely, therefore, that modifications in the clonal compositions of available versus actual repertoires imposed by priming set in motion regulatory mechanisms which are independent of the inducing ligand. Long after priming, the immune system of these animals retains a ~memory>> of that experience reflected by new rules in ongoing repertoire selection. In the example given here, this memory is essentially ~ negative ~, leading to unresponsiveness. The basis for this phenomenon therefore appears to be the der, letion of small B cells with relevant specificities. In turn, such depletion is not compensated for by the increase in the same reactivities in the acti',ated, large B-cell pool. This is already the case 5 days after priming, as we had previously shown [6], and it probably reflects inauction of specific B cells to nondividing, Ig-secreting cells by Dex or anti-Id-LPS in the absence of marked clonal expansion. With persisting antigen, the same mechanism could be invoked to continuously deplete specific cells from the incoming newly formed small B lymphocytes. This would imply, however, sustained antibody resr onses which, even after Dex-priming, are not detected. Alternatively, persistent multivalent ligands could possibly inactivate newly formed B cells in the same manner in the absence of their terminal maturation. Finally, in cases in which persistence of the inducing ligand cannot be invoked, it is plausible that newly formed B cells are continuously activated (by Id-specific mechanisms) into a compartment of large, V-region-connected cells that turn over rapidly [17], mount pool' immune responses and are inhibited in Ig secretion by mechanisms related to ~ anti-~ inhibition >>precisely because of idiotypic interactions. In fact, even in normal mice, only around 5-10 % of all specific, naturally activated cells secrete enough antibody to be detected as a PFC, although they all do if stimulated by a mitogen in LDA, when they are no longer submitted to such receptor-mediated network interactions.

REFERENCES [1] LUNDKVIST,I., IVARS,F., HOLMBERG,D. & COUTINHO,A., The immune response to bacterial dextrans. -- V. A ~ dominant)) idiotype in IgCH b mice. J. Immunol., 1987, 138, 4395-4401. [2] LUND~VIST,I., PORTNOI,D. ~ COUTINHO,A., The immune response to bacterial dextrans. -- Vl. No correlation between the frequency of cells expressing a major anti-dextran idiotype and the idiotype profiles of specific antibody responses. Scand. J. Immunol. (in press). [3] PORTNOI, D., LUNDKVIST,I. 8/: COUTINHO, A., Inverse correlation between the utilization of an idiotype in specific immune responses and its representation in pre-immune ~ natural ~) antibodies. Europ. J. lmmunoL, 1988, 18, 571-576.

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[4] FERNANDEZ,C. • MOLLER,G., Antigen-induced strain-specific autoantiidiotypic antibodies modulate the immune response to dextran B512. Proc. nat. Acad. Sci. (Wash.), 1979, 76, 5944-5947. [5] CotrrlmtO, A., FORNI, L., HOLMBERG, D., IVARS, F. & VAZ, N., From an antigen-centered clonal perspective of immune responses to an organismcentered network perspective of autonomous activity in a self-referential immune system. Immunol. Rev., 1984, 79, 151-168. [6] PORTSOI, D., LUNDKVlST,I. & COUTINHO, A., On the validity of LPS-driven limiting dilution systems for clonal B-cell precursors to analyse functional antibody repertoires. Scand. J. Immunol., 1988, 27, 445-450. [7] ANDEmSSON,J., COUTINHO,A., LEm~HARDT,W. & MELCHERS,F., Clonal growth and maturation to immunoglobulin secretion in vitro of every growth inducible B lymphocyte. Cell, 1977, 10, 27-34. [8] NATnANSON, S.D., ZAMFmESCU, P.L., DREw, P.I. & WlLBUi~, J., Two-step separation of human peripheral blood monocytes in discontinuous density gradients of cC3oidal s~ca-polyvinyl pyrrodine. J. lmmunol. Methods, 1977, 18, 225-234. [9] Lta~aST, I., HOLMBERG,D., IVAI~, F. & Cotrnmto, A., The immune response to bacterial dextrans, m III. Ontogenic development and strain distribution of specific clonal precursors. Europ. J. lmmunol., 1986, 16, 957-962. [10] G~mmVOOD, F.C., HUNTER, W.M. & GLOVER, J.S., The preparation of 12SI-labelled human growth hormone of high specific radioactivity. Biochem. J., 1963, 89, 114-123. [11] HOWARD, J.G., Cellular events in the induction and loss of tolerance to pneumococcal polysaccharides. Transplant. Rev., 1972, 8, 50-75. [12] MIRANDA,J.J., ZOLA,H. & HOWARD,J.O., Studies on immunological paralysis. X. Cellular characteristics of the induction and loss of tolerance to levan (polyfructose). Immunology, 1972, 23, 843-855. [13] DRtrrz, D.J. & GRAYBILL,J.R., Infectious diseases, in <