xid mice fail to express an anti-dextran immune response but carry alpha(1-3)dextran-specific lymphocytes in their potential repertoire

Even after repeated immunizations with alpha(1-3)dextran (Dex) male (CBA/N x BALB/c)F1 mice fail to produce specific antibodies whereas nondefective female littermates express an idiotype-positive anti-Dex immune response. This failure of xid mice to express serum anti-Dex immunoglobulins is not only limited to immunization with the thymus-independent (TI-2) antigen Dex, since immunization with anti-idiotypic antibodies against Dex-specific idiotypes does not overcome this defect. When xid lymphocytes are cultured in the presence of mitogens, in vitro anti-Dex responses are markedly reduced. We show here that alpha(1-3)Dex-specific hybridomas can be established by fusion of splenic lymphocytes from xid mice to Sp2/0 myeloma cells. Therefore, these mice do carry the potential to generate B cells specific for Dex. All hybridoma antibodies were found to be of IgM isotype, bearing the lambda light chain typical for alpha(1-3)Dex-specific antibodies. Whereas monoclonal anti-Dex antibodies obtained from spleen cell hybridomas from female littermates showed variable idiotope patterns, hybridoma proteins from the immune-defective NBF1-xid mouse expressed only a limited pattern of Dex-specific idiotopes, suggesting that these hybridomas derived from a common precursor.     

Stimulation of the Same B-Cell Population by Thymus-Independent Dextran and by Thymus-Dependent Oligosaccharide-Carrier

The immune response of adult BALB/c mice against the bacterial antigen dextran B13558 (Dex) is well characterized as thymus independent (TI type 2) and Igh^a linked. The antisera consist of mainly IgM/ antibodies directed against the λ(1 → 3) glucosidic linkage. This study describes the immune reponse against the α(1 → 3) linkage in the thymus dependent form (TD), i. e. tetra- or heptasaccharides (N4 or N7) of glucose as hapten coupled to chicken serum albumin (CSA) as carrier. Whereas athymic BALB/c-nu/nu mice did not respond to the TD antigens N4-CSA and N7-CSA, euthymic BALB/c showed high anti-Dex antibody titres of IgM and, after 2° immunization, a class switch to IgG (mainly IgGl) isotypes with λ light chains. The hapten N4 inhibited Dex-binding of M104E or of antisera from Dex or N4-CSA or N7-CSA immunized mice at 1.7–10 × 10⁴M. The idiotype composition of these antibodies resembled those after Dex immunization. We conclude that the same Dex-specific precursor B cells have been stimulated by either form of antigen. The ontogenic development of a Dex-specific response could not be accelerated by the aid of T cells, even of adult origin. It seems, therefore, that the maturation of antigen specific B cells is the limiting step in ontogeny.     

Nude mice are nonpermissive towards the anti-dextran response of congenic cells

Nude mice bearing allotype Ighb on a BALB/c genetic background (= CB nu/nu) are nonresponders to alpha (1----3)dextran (Dex), in contrast to BALB/c or BALB/c nu/nu. Although CB nu/nu mice accept transplants of congenic BALB/c, or BALB nu/nu lymphocytes, as shown by the expression of donor allotype Igha, they are not permissive for a primary anti-Dex response by the grafted cells. BALB/c or BALB nu/nu cells, however, give a strong anti-Dex response when grafted onto irradiated CB nu/nu or CB 23 (Ighb) euthymic mice. A thymus-independent, radiation-sensitive suppressor cell population is postulated, which specifically hinders the anti-Dex response, and which is exhibited by strains bearing that portion of chromosome 12 which codes for CH allotype Ighb, not containing the germ-line anti-Dex V/D genes. The suppressive action of Ighb lymphocytes could be demonstrated directly in staggered co-transfer experiments.     

Immune response against the T-independent antigen alpha (1 leads to 3) dextran. I. Demonstration of an unexpected IgG response of athymic and germ-free-raised euthymic BALB/c mice

The primary antibody response in BALB/c mice to the T-independent bacterial antigen dextran B1355S [alpha(1 leads to 3)dextran] (Dex) was studied by means of isoelectric focusing, hemagglutination and immunodiffusion techniques. In response to a single immunization with 10 micrograms Dex all mice produce specific IgM antibodies. In addition, about 30% of conventionally raised BALB/c and BALB/c nu/ + mice, but 95% of germ-free (GF)-raised normal BALB/c and 100% of athymic BALB/c nu/nu mice produce specific IgG class anti-Dex antibodies. These antibodies include all IgG subclasses, carry predominantly the lambda light chain and the cross-reactive J558 idiotype and are specific for the alpha(1 leads to 3)glucosidic linkage. As compared to athymic and GF-raised mice, conventionally raised mice exhibit only a weak IgG response. The pronounced IgG production of GF-raised mice was not altered when adult mice were removed from their GF environment and housed under conventional conditions for several weeks prior to immunization with Dex. Reconstitution with isolated splenic T cells from conventionally raised, unprimed BALB/c mice reduces the remarkable capacity of BALB/c nu/nu mice to produce IgG anti-Dex antibodies. These findings suggest that the reduced capacity of conventionally raised BALB/c mice to mount an IgG response to the T-independent antigen Dex is due to a T cell-mediated suppressive mechanism which is neonatally induced by contact with environmental, i.e. bacterial, antigens.     

Involvement of CD28 cosignalling in the T cell-mediated suppression of the IgG antibody response against the TI-2 antigen alpha(1-->3) dextran.

The humoral immune response against alpha(1-->3) dextran (Dex) in BALB/c mice is characterized by the formation of predominantly IgM antibodies bearing the J558 idiotype. IgG antibodies do not appear in euthymic mice. In athymic animals, however, the response proceeds to a vigorous IgG production. In euthymic mice formation of IgG is suppressed by J558 idiotype specific regulatory T cells recognizing in association with I-Ed and in cognate T/B interaction the V(H) CDR3 derived peptide of the J558 idiotype. Only B-2 lymphocytes produce IgG whereas B-1 cells do not participate in the production of this Ig class. Using novel synthetic all alpha(1-->3)-D-gluco configured tetrasaccharide the Dex-specific B cells can for the first time be analyzed in FACS. In experiments using this newly designed low molecular Dex no signs of B cell apoptosis can be found. This demonstrates a true silencing of persisting Bgamma memory cells as previously suggested by adoptive transfer experiments. In this suppression a further involvement of CD28 and B7-1 interaction can be demonstrated which delivers a necessary costimulatory suppression signal in addition to the cognate TCR/peptide-I-Ed interaction between J558 specific T cells and J558 idiotype bearing B cells.     

The immune response to bacterial dextrans. III. Ontogenic development and strain distribution of specific clonal precursors

The frequencies of B512 dextran (Dex)-specific B cell precursors were determined by limiting dilution analysis in a number of mouse strains originally described as "high responder", "low responder" and "nonresponder" to this antigen. No significant difference in the frequencies of Dex-specific precursors was found in C57BL/6, B10.BR, C3H/Tif, BALB/c and A/Sn adult mice. Together with the large intra-strain variability in the magnitude of anti-Dex PFC responses in vivo, these results established that differential reactivity in vivo cannot be ascribed to genetically controlled absence or wide variation in the frequency of Dex-specific immunocompetent precursors. A similar analysis of the Dex-specific precursor frequency was carried out in C57BL/6 mice between 1 week and 3 months of age. While no Dex-specific antibody response was detected in vivo before the age of 3 weeks, clonal precursor analysis revealed that the appearance of these specificities parallels the development of competent (IgM-producing) B lymphocyte clonal precursors, such that no significant difference in absolute frequencies of Dex-specific precursors could be observed among these age groups. This is interpreted to suggest that the late development of the Dex-specific antibody responses is regulatory rather than due to late rearrangement and activation of the appropriate V genes and a sequential expression of antibody specificities in ontogenic development.     

Thymus-independent induction of idiotype suppression in newborn mice by syngeneic anti-idiotype antisera

BALB/c and BALB/c nu/nu mice were shown to express to a variable extent in their response against dextran B1355S (Dex), an idiotype which is present on the Dex-reactive BALB/c myeloma protein MOPC 104E. Injection of minute amounts of syngeneic anti-MOPC 104E idiotype antisera into neonatal euthymic or athymic BALB/c mice suppressed this idiotype in the Dex-specific response of the adult animals. When spleen cells from suppressed BALB/c mice were transferred into irradiated BALB Ighb mice the state of suppression persisted. Data are discussed with respect to possible mechanisms regulating expression of this idiotype.     

The J558 V(H) CDR3 region contributes little to antibody avidity; however, it is the recognition element for cognate T cell control of the alpha(1-->3) dextran-specific antibody response [In Process Citation]

Analysis of the humoral immune response of BALB/c mice to alpha(1-->3) dextran (Dex) reveals novel aspects of T cell-mediated control of 'type 2 thymus-independent' responses against polysaccharide antigens. The IgM and IgG antibody response, dominated by the J558 idiotype (Id), is controlled by Id-specific T cells. These regulatory T cells, for which the T cell clone 178-4 Ts with characterized TCR alpha and beta chain sequences is the prototype, expand in all BALB/c mice upon immunization with Dex. They suppress in a cognate interaction the expansion of J558 Id-bearing B cells, committed for production of IgG antibodies. Furthermore they provide a gate which precludes variability in the VH CDR3 region of IgG antibodies appearing occasionally in the periphery. The VH CDR3 region is the recognition element of 178-4 Ts analogous T cells but contributes little to affinity for the antigen. For recognition by 178-4 Ts cells not even minimal sequence deviations of the J558 Id peptide are allowed. The tight germline programmed complementarity between J558 Id-bearing Dex-specific B and J558 Id- specific 178-4 Ts analogous T cells leaves little room on both sides for ontogenetic variability.      

The thymus-independent antigen α(1–3) dextran elicits proliferation of precursors for specific IgM antibody-producing cells (memory cells), which are revealed by LPS stimulation in soft agar cultures and detected by immunoblot

Single antibody-forming cells (AFC) specific for α(l-3) dextran (Dex) from i.p.-immunized BALB/c mice were enumerated in soft agar cultures by blotting on antigen-precoated membranes and subsequent staining via enzyme-coupled anti-IgM antibodies. Short cultures (2 h) revealed AFC as harvested ex vivo, while in long-term cultures (4 days), in the presence of lipopolysaccharide (LPS) as B cell mitogen, cells or colonies developed by differentiation in vitro. Whereas the spleen contained most AFC ex vivo in a sharp-peak response at 4 and 5 days after i.p. injection of Dex in aqueous solution, peritoneal exudate cells (PEC) contained only very few AFC. However, the same PEC population developed Dex-specific cells or colonies after 4 days of culture. The isotype of antibodies was IgM. The frequency of these Dex-specific LPS-inducible precursor cells rose exponentially in the course of the immune response to a broad plateau and was still, 11 weeks after Dex injection, approximately 40-fold higher than in non-immunized mice. Since these cells increased in frequency after antigen injection, and since they could not be detected as AFC during 2 h ex vivo, they were regarded as memory cells. They seemed to be arrested in vivo, but could be induced to differentiation and/or proliferation in vitro. Although these cells had the functional characteristics of memory cells as defined above, they produced anti-Dex antibodies of IgM isotype. Their population might be critical for the protection of the peritoneal cavity against microbial invasion from the intestines, and it may be significant in this context that we could evoke a peritoneal memory cell response only when antigen was injected intraperitoneally, but not intravenously. In athymic BALB/c-nu/nu mice only few of these Dex-specific memory cells were found. It is possible that T cells exert a regulatory influence on this pathway of differentiation.     

The Immune Response to Bacterial Dextrans

C57BL/6 mice respond to Dextran B512 (Dex) with a predominant idiotype (Id) (17-9) while no such Id-positive antibodies are identified in the specific antibody response of BALB/c mice. We used limiting dilution systems to determine the absolute frequencies of clonal B-cell precursors producing the 17-9 Id in these two mouse strains and analysed the correlation between Id-expression and antibody activity at the clonal level. The results show very similar frequencies of anti-Dex and Id-positive B cells in both strains, but C57BL/6 mice contained fourfold higher frequencies of Dex-specific clonal precursors which are Id-positive. This kind of clone, although not used in the specific response of BALB/c mice, constitute roughly 20% of their anti-Dex repertoire and they are readily induced in this strain. Thus, immunization of both strains with anti-idiotypic antibodies results in the production of Id-positive anti-Dex antibodies with serum titres that directly correlate with precursor frequencies. The results show, therefore, that the section of clonal repertoires utilized in a primary immune response varies with the immunogen, even if thymus-independent. These observations are discussed in the context of the genetic controls of anti-Dex antibody responses and on the general question of the utilization of available antibody repertoires in immune responses.     

Idiotype-specific CD4+CD25+ T suppressor cells prevent,by limiting antibody diversity,the occurrence of anti-dextran antibodies crossreacting with histone H3

CD25(+) suppressor T cells regulate the immune response against the type-2 "thymus independent" bacterial polysaccharide antigen alpha(1-->3)dextran (Dex) in BALB/c mice. These T cells, represented by the clone 178-4 Ts, restrict the Dex-specific IgG antibody repertoire such that the J558 idiotype dominates. Antibodies with other structures in the heavy-chain variable region (V(H) region), predominantly within the CDR3 domain, occur when the T cell control fails. This increase of antibody diversity caused by a lack of CD25(+) Ts cells, e.g. in nude mice, does not result in the appearance of antibodies with enhanced affinity to the antigen Dex, but often leads to a crossreactivity with autologous proteins. Twenty-two out of sixty Dex-specific hybridomas from nude mice, but no hybridomas from euthymic mice, crossreact with a nuclear protein, as tested by ELISA. This nuclear protein was identified as histone H3. Ten of the sixty hybridomas from nude mice were sequenced and show V(H) sequences that deviate from the original J558 sequence. Three of these ten hybridomas crossreact with the histone H3. Adoptive transfer of CD25(+) Ts cells to nude mice leads to a marked increase of antibodies carrying the original J558 idiotype within the IgG pool after immunization with Dex. Our data demonstrate a CD25(+) Ts cell-mediated restriction of V(H) usage, which prevents the appearance of crossreactive autoantibodies.     

CD3+ T-cells in severe combined immunodeficiency (scid) mice. III. Transferred congenic, selfreactive CD4+ T cell clones rescue IgM-producing, scid-derived B cells.

Young severe combined immunodeficiency (scid) mice completely lack immunocompetent lymphocytes. Limiting numbers of purified CD4+ T cells from allotype-congenic BALB/c (Igha) donor mice were transplanted into 3-week-old scid (Ighb) recipient mice. Splenic CD4+ T cells were recovered from transplanted scid mice 10-12 weeks post-transfer and established as T cell lines in culture. These T cell populations proliferated in vitro in response to syngeneic stimulator cells. T cell clones derived in vitro from these T cell lines displayed selfreactive recognition specificity: these CD4+ T cells proliferated in vitro in response to syngeneic/congeneic but not allogeneic stimulator cells. Cloned selfreactive T cells retransplanted into young scid recipients were engrafted into spleens of secondary recipients, did not induce autoimmune disease but stimulated development of scid-derived (Ighb), IgM-producing B cells (B cell leakiness).     

Cremophor EL as an adjuvant affecting immunoglobulin class switch in the immune response to the thymus-independent antigen alpha(1- greater than 3) dextran B 1355 S.

The humoral immune response to the so-called thymus independent antigen dextran B 1355 S in conventionally raised BALB/c mice consists solely of IgM antibodies. Expression of IgG anti-Dex antibodies in these mice is prevented by pre- or perinatally activated idiotype-specific T-suppressor lymphocytes. IgG B-memory cells nevertheless develop during the course of immunization, but are arrested in an anergic state. In the presence of Cremophor EL the induction of this anergic state is inhibited and the immune response shifts fully to an IgG anti-Dex response.     

Inverse correlation between the utilization of an idiotype in specific immune responses and its representation in pre-immune "natural" antibodies

Previously we have characterized an idiotype (Id) that accounts for half of all specific anti-dextran B512 (Dex) antibodies in C57BL/6 mice. BALB/c mice produce the same Id in normal, pre-immune sera but fail to use it in antibody responses to Dex, although Id+ anti-Dex antibodies can be induced in this strain by anti-Id immunization. By limiting dilution analysis of B cell clonal precursors, we show here that the frequencies of Id+ B cells are comparable in both strains, but their state of activity is sharply distinct: while all Id+ B cells are small, resting lymphocytes in C57BL/6 mice, they are all large, naturally activated cells in BALB/c mice. The suggestion that naturally activated cells are poorly engaged in specific responses was supported by the delayed and lower Id+ responses obtained in BALB/c mice when they are immunized, in parallel with C57BL/6 animals, with a conjugate of anti-Id antibodies and lipopolysaccharide. Finally, C57BL/6 responder mice were found to closely reproduce the normal BALB/c situation, if analyzed 3 months after anti-Id priming: they produce low levels of serum Id and all Id+ B cells are in the large lymphocyte compartment. Upon immunization these animals develop serum Id+ responses that are undistinguishable from low-responder BALB/c mice. The relevance of these observations for the questions of physiologic self-reactivity is discussed.     

Lymphokine profile and activation pattern of two unrelated antigen- or idiotype-specific T suppressor cell clones.

Two T suppressor (Ts) clones of different specificity have been analyzed for their lymphokine spectrum. BVI/5 is an I-Ek-restricted bovine serum albumin (BSA)-specific Ts cell clone from a CBA/J mouse tolerized by low doses of BSA. It affects directly or indirectly the function of BSA-specific T helper (Th) cells. The Ts cell clone 178-4 from a BALB/c mouse is I-Ed restricted and recognizes the public J558 Id on B cells. It prevents alpha(1----3)dextran B 1355S (Dex)-specific IgG antibody production and drives Dex-specific J558 idiotype-bearing B cells into an anergic B IgG memory cell state. Both Ts cell clones thus cause specific suppression, yet in different experimental systems using different effector mechanisms. Upon stimulation with concanavalin A or fixed CD3-specific monoclonal antibody, both clones produce high levels of interferon (IFN)-gamma and tumor necrosis factor (TNF) but in contrast to Th1 cells no interleukin (IL)-2. Both clones produce low levels of IL-3 and IL-6 but no IL-4, IL-5 and IL-9. Furthermore, unlike Th2 cells, both clones do not respond to IL-1. The mechanism of the idiotype-specific induction of anergy in Dex-specific B IgG memory cells by 178-4 Ts cells is not yet understood. BVI/5 Ts cells suppress in vitro the BSA-specific proliferation of the BSA-specific Th cell clone 83/1, as well as the response of BSA-primed CBA/LN cells. Whereas the suppressive effect on 83/1 cells is due to IFN-gamma alone the suppression of BSA-specific lymph node cells can be simulated neither by IFN-gamma nor the combination of IFN-gamma and TNF. Thus these mediators cannot account for the antigen-specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from lymph node cells and probably not for the induction of in vivo unresponsiveness.     

A myeloma M 104E private idiotope is represented predominantly among anti-dextran, peritoneal cavity Ly-1+ precursor B cells.

Public and private idiotopes (Id) had been defined in the BALB/c anti-dextran B1355 fraction S (Dex) response by means of syngeneic monoclonal anti-M 104E Id antibodies. Most of the primary anti-Dex antibodies shared the public Id-1 while the private Id-5 constituted a comparatively low, but highly variable proportion of humoral anti-Dex antibodies of individual mice. In the present work we have analyzed by limiting dilution the expression of these two Id by anti-Dex B cell precursors from the spleen and peritoneal cavity of BALB/c mice. Testing spleen cells we found about 65% of anti-Dex precursors to be Id-1+ and only a minority to be Id-5+. Treatment with anti-Ly-1 and complement was without any effect on anti-Dex precursors from spleen. Anti-Dex B cell precursors among peritoneal cavity lymphocytes differed from those found in the spleen in two ways. First, on average, 50% of them were Id-5+; second, about 80% carried the Ly-1 marker. Both markers are, therefore, expressed on at least 30% of anti-Dex B cell precursors from the peritoneal cavity but rarely on anti-Dex precursors from the spleen.     

Physiology of IgD. III. Effect of treatment with anti-IgD from birth on the magnitude and isotype distribution of the immune response in the spleen

Continued treatment with monoclonal anti-IgD (Ig-5a) from birth in BALB/c mice causes a markedly increased responsiveness to i.v. injected dinitrophenylated ovalbumin (DNP-OVA) with Bordetella pertussis at the age of 8 weeks. The 19S plaque-forming cell (PFC)/spleen response is particularly enhanced, 6-8-fold, but all the other isotypes also show increases of 2-6-fold, including IgA and IgE. Both primary and secondary PFC responses and serum antibody titers are enhanced. After transfer of spleen cells from anti-Ig-treated mice to irradiated recipients the IgM/IgG ratio becomes similar to that of controls. In contrast, the response of anti-IgD-treated mice to i.p. immunization with either 0.2 or 100 micrograms DNP-OVA plus alum is reduced by approximately 80% for each Ig isotype except IgM and remains low upon transfer of spleen cells to recipients. It is concluded that the paucity of B cells in peripheral lymph nodes of the anti-IgD-treated mice causes the low responsiveness to i.p. immunization, but that the IgD- B cells in the spleen are quite able to respond and are, in fact, more responsive than IgD+ B cells. This increased responsiveness, together with the higher IgM/IgG ratios for all Ig isotypes and an otherwise similar order of isotype distribution (gamma 1 greater than gamma 2b greater than gamma 2a = epsilon greater than or equal to alpha) as in controls, suggests that a hyperresponsive, but less mature IgD- B cell population is selectively produced in the spleens of mice treated with anti-IgD from birth.     

Normal T splenocytes are able to induce immunoglobulin allotypic suppression in F1 hybrid mice

A chronic suppression of Igh-1b and Igh-3b (IgG2a and IgG2b of b haplotype) allotype expression has been induced by injecting T splenocytes from normal BALB/c or BC8 mice into newborn F1 hybrids of appropriate Igh congenic strains: BALB/c into (BALB/c Igha X CB20 Ighb)F1 and BC8 into (BC8 Igha X C57BL/6 Ighb)F1 or (C57BL/6 X BC8)F1. This suppression does not affect IgM (IgH-6b) or IgA (Igh-2b) expression. When the Ighb haplotype is paternally transmitted, the proportion of T splenocyte recipients showing allotypic suppression increases with time reaching 70% 40 weeks after birth. We also succeeded in inducing this pattern of suppression in 2 out of 13 cases when the Ighb was inherited from the mother. These normal T splenocytes are therefore clearly allotype specific. As Igh-6b production is not affected by the suppression, these T splenocytes are believed to influence B cells more or less committed to Igh-1b or Igh-3b production rather than more precocious Igh-6b (IgM of b haplotype) carrying precursors in the classical IgM-IgG filiation pathway.     

Idiotype suppression by maternal influence.

In BALB/c mice, antibodies to the alpha-(1-3) glucosidic linkage of some dextrans (Dex) carry the idiotype of the BALB/c myeloma protein J558. Both specific antibody and idiotype are inherited in a dominant fashion, linked to the immunoglobulin (Ig) (heavy chain) allotype Igla of BALB/c mice (Eur. J. Immunol. 1975. 5: 775). In F1 hybrid mice from the parent strains SJL and BALB/c, we were able to suppress the expression of anti-Dex antibodies by immunizing prospective SJL mothers to the J558 idiotype. The state of suppression in the progeny was ascertained by immunization with Dex, and tests for the following were carried out: (a) antibodies specific for Dex; (b) inhibition of such antibodies (if present) by antiidiotypic serum to protein J558; (c) presence of the J558 idiotype; and (d) concentration of lambda1 chains (which are associated with the 558 idiotype) in the serum. SJL mothers, once immunized, conferred suppression upon several successive litters, spanning a period of 4-5 months. Suppression in F1 progeny animals lasted for 16 weeks or more. Spleen cells from suppressed F1 mice which had neither been treated with Dex nor with J558 protein, were able to confer suppression to further F1 newborn mice.     

Isogeneic monoclonal antibodies against anti-alpha(1----3)dextran idiotypes. I. Isotypes, idiotope specificity and representation of idiotopes in antisera from mice of various genetic constitutions

Eight isogeneic anti-idiotypic hybridomas were raised against BALB/c myeloma protein MOPC 104E and one against J558. Both myelomas react specifically with the alpha(1----3) glucosidic linkage of dextran B1355 fraction S (Dex). Six anti-MOPC 104E proteins were IgG1, one was IgG2b and one IgM. The anti-J558 protein was IgG1. Competitive interactions of the anti-idiotopes and antigen with anti-Dex proteins were measured. Dex itself was effective, but also an alpha(1----3) glucosidic heptasaccharide (N7-CHO). In order to assess the anti-idiotope specificity of hybridoma proteins, three anti-Dex molecules were used: MOPC 104E, J558 and hybridoma protein Hdex14. These differed from each other in VH amino acid positions 54-55, or 100-101, respectively. By their serological reaction pattern our anti-idiotope proteins could be divided into 3 groups: cross-reactive, partially cross-reactive and strictly specific for the immunogen. The latter ones were in the majority, and were called "private", in contrast to the cross-reactive "public" anti-idiotopes. The serological pattern was followed, in general, by the mouse-to-mouse distribution of idiotopes in physiological anti-Dex sera. Public idiotopes were closely correlated in their expression with anti-Dex activity. "Private" idiotopes showed no correlation, and displayed a characteristically high degree of fluctuation from mouse to mouse. Among the different mouse strains that were compared with respect to idiotope expression in anti-Dex sera, two stand out: C57BL-Igha, which carries chromosome 12 of BALB/c, (as selected through allotype) on the C57BL/6 genome, and BALB-Ighb, dex+, a recombinant in chromosome 12 linking the dex+ trait from BALB/c to the CH allotype from C57BL/6. The latter strain expressed significantly more of the private idiotopes than the former. This observation is discussed in terms of the position effect of classical genetics and network concepts.