Idiopathic paraproteinaemia. IV. The role of genetic factors in the development of monoclonal B cell proliferative disorders--a study in the ageing C57BL/KaLwRij and CBA/BrARij mouse radiation chimeras

Mouse radiation chimeras, employing strains with a low (CBA/BrARij) and a high (C57BL/KaLwRij) frequency of idiopathic paraproteinaemia (IP), were used in a study on genetic influences in the development of IP, a benign B cell monoclonal proliferative disorder. Taking advantage of the different Igh1 allotypic markers between the two strains, the development of IP with increasing age was investigated by agar electrophoresis, immunoelectrophoresis and immunofixation. Four of 18 CBA recipients transplanted with C57BL bone marrow cells were shown to develop IP of the IgG2a isotype and the Igh1b (donor) allotype during their life. In contrast, none of the 23 C57BL recipients of CBA bone marrow developed an IgG2a paraprotein of the Igh1a allotype. However, in three of these 23 chimeras, an IgG2a and Igh1b (recipient) allotype paraprotein appeared with age; two of these mice proved to be reversals at 12 months and one at 15 months of age. The frequencies of homogeneous immunoglobulins of the donor type in the chimeras corresponded roughly to those of normal mice of the donor strain. Histopathological examination excluded a malignant origin of these monoclonal proliferations. These findings support the view that intrinsic cellular genetic factors are of major importance in the development of IP, a benign B cell neoplasia.     

The influence of genetic factors associated with the immunoglobulin heavy chain locus on the development of benign monoclonal gammapathy in ageing IgH-congenic mice.

The role of genetic factors associated with the immunoglobulin heavy chain locus (Igh) in the development of benign monoclonal gammapathy (BMG), a benign B-cell proliferative disorder, was investigated in six Igh congenic mouse strains during ageing. The strains used had a C57BL or BALB background: C57BL/6, BALB.Igb and CB-20 carrying the C57BL Igh (Ighb allotype), BALB/c and C57BL/6.Iga carrying the BALB/c Igh (Igha allotype) and BAB-14, that is of BALB/c origin with the exception of the constant part of the Igh, which is of C57BL origin. The frequency of homogeneous immunoglobulins (H-Ig), both single and multiple, was the highest in C57BL/6 mice, followed by C57BL/6.Iga. The frequencies of H-Ig in BALB.Igb and CB-20 mice were higher than those of BALB/c and BAB-14, although somewhat lower than in C57BL/6.Iga mice. Multiple H-Ig were found especially in the sera of C57BL/6 mice. Categorization of the monoclonal gammapathies (MG) on the basis of their origin showed a single transient monoclonal B-cell proliferation in 0-8% of the mice of all strains. Persistent, non-progressive MG, presumably BMG, were detected in 64% of C57BL/6, 30% of C57BL/6.Iga, 22% of BALB.Igb, 17% of CB-20, 13% of BAB-14 and 6% of BALB/c mice. Multiple myeloma or Waldenström-like B-cell lymphoma were found to be responsible for 2-4% of the paraproteinemias in all strains. The remaining H-Ig, varying from 11% of the C57BL/6 to 70% of the BAB-14 mice, could not be evaluated in time. The most frequent isotypes of the BMG within C57BL/6 and C57BL/6.Iga were IgG2a and IgG2b, respectively; IgM was the most frequent isotype within the four BALB congenic strains. The immunoglobulin heavy chain allotypes under investigation appeared to be only partly related to the onset, occurrence, multiplicity and persistence of the BMG developing in these Igh congenic C57BL and BALB strains during ageing. The immunoglobulin heavy chain allotypes, however, were not related to the major isotype of the BMG. The results obtained in CB-20 and BALB.Igb on the one hand, and in BAB-14 on the other hand, may suggest a role for the variable part of the Igh in the development of BMG. Since no absolute influence could be ascribed to the Igh, we assume that primarily other genetic sequences regulating proliferative B-cell functions account for the pathogenesis of BMG.     

The influence of H-2 genetic factors on the development of benign monoclonal gammopathy in ageing H-2 congenic C57BL and BALB mice.

The role of H-2 genetic factors in the development of benign monoclonal gammopathy (BMG) was investigated in six H-2 congenic C57BL and BALB strains (C57BL/10.ScSn and BALB.B: H-2b; B10.D2 and BALB/c: H-2d; B10.BR and BALB.K: H-2k) during ageing. The frequencies of homogeneous immunoglobulins (H-Ig), both single and multiple, in the three C57BL strains were higher than those in the corresponding three BALB strains. No relationship was found with a particular H-2 haplotype. The most frequent H-Ig isotype within the C57BL strains was IgG2a, within BALB.B and BALB.K mice IgG3 and in BALB/c mice IgG1. Categorization of the monoclonal gammopathies (MG) on the basis of their origin showed a single transient monoclonal B-cell proliferation in 2-5% and 3-9% of the C57BL and BALB mice positive for H-Ig, respectively. Multiple myeloma or B-cell lymphoma were found to be responsible for about 1% of the paraproteinaemias in all strains. Persistent, non-progressive MG, most likely BMG, was detected in 70-81% and 39-46% of the C57BL and BALB mice positive for H-Ig, respectively. The remaining 14-24% and 50-58% of the, respectively, C57BL and BALB mice positive for H-Ig could not be evaluated in time. The H-2 haplotypes under investigation were not associated with the onset, occurrence, multiplicity, persistence or isotype of the MG developing in these H-2 congenic C57BL and BALB strains during ageing.     

Identification of novel VH1/J558 immunoglobulin germline genes of C57BL/6 (Igh b) allotype

Although a rich database of Igh a allotype mouse immunoglobulin germline genes exists, current information on Igh b allotype immunoglobulin germline genes is limited. Among the immunoglobulin VH genes, single-cell amplified from six Igh b (C57BL/6 background) spleens in this study, 602 clonally independent immunoglobulin VH sequences belonging to the VH1/J558 family were identified. Whereas 335 of these sequences could be traced to have originated from 29 different VH1/J558 germline genes deposited in the NCBI Igblast database, the remaining 267 sequences appeared to have originated from 21 novel germline genes. Of the 50 VH1/J558 germline genes utilized in the peripheral repertoire of these Igh b allotype mice, the most frequently used genes included 45.21.2, V165.1, J558.6, J558.18A, and V23. Whereas the majority of the novel genes uncovered represented allelic counterparts of previously described Balb/c (Igh a allotype) genes, some appeared to represent truly novel germline genes. Collectively, the VH1/J558 germline genes exhibited high amino acid residue usage variability at the CDR1 positions, H31, H33, and H35, and the CDR2 positions, H50, H52, H53, H54, H56, and H58. The 50 VH1/J558 germline genes expressed in the peripheral Igh b repertoire also varied widely in the net charge of their CDR regions, raising the possibility that they may be differentially utilized to encode anti-nuclear autoantibodies.     

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.     

Genetic Analysis of the Low Responsiveness to Dextran B512 in CB A/N and C57BL/10ScCr Mice

CBA/N and C57BL/10ScCr mice are low responders to the antigen dextran B512. This is due to the Xid gene in CBA/N mice and to unknown genes in C57BL/10ScCr mice, although this strain is unresponsive to lipopolysaccharide (LPS) due to a defective gene in the fourth chromosome. The female F1 hybrids (C57BL/10ScCr X CBA/N) and (CBA/N X C57BL/10ScCr) were low responders to dextran, although the Xid gene is not expressed in these hybrids, indicating lack of genetic complementation. In contrast, female F1 hybrids between the dextran high-responder strains CBA or C57BL/10 as one parental strain and the low-responder strains CBA/N or C57BL/10ScCr as the other parental strain, respectively, were responders to dextran. The C57BL/10ScCr mice did not appear to have an X-linked gene determining low responsiveness to dextran. The findings suggest that the only defect in CBA/N mice cannot be the Xid gene and the only defect in C57BL/10ScCr mice cannot be the gene determining unresponsiveness to LPS.     

Effects of IgM Allotype Suppression on Serum IgM Levels,B-1 and B-2 Cells,and Antibody Responses ir Allotype Heterozygous F1 Mice

IgM allotype heterozygous F1 mice were independently suppressed for Igh6a or Igh6b to evaluate the contribution of B-1 and B-2 cells to natural serum IgM levels and Ab responses. B-2 B cells expressing IgM of the suppressed allotype were evident in the spleens of suppressed mice 4 to 6 weeks after cessation of the suppression regimen, whereas B-1 B cells of the suppressed allotype were undetectable for up to 9 months. Although serum IgM of the suppressed allotype was initially depleted in mice suppressed for either allotype, by 7 months of age, there were detectable levels of IgM of the suppressed allotype in the serum; however, the levels were significantly below that found in nonsuppressed mice. When mice were immunized with either the T-independent or T-dependent form of phosphorylcholine, those suppressed for either allotype, and consequently depleted of B-1 B cells of that allotype, did not respond with phosphorylcholine-specific IgM of the suppressed allotype. In contrast, when mice were immunized with α1-3 dextran, the Igh6a allotype-suppressed mice were able to produce dextran-specific IgM of that allotype. These results show that allotype-bearing B-1 cells of both allotypes can be effectively suppressed by this suppression protocol and this produces long-lasting effects on B-1 cell levels and serum IgM of the suppressed allotype. These observations reflect the derivation of the majority of B-1 cells from fetal-neonatal precursors, which cannot be replaced by newly emerging B-2 cells of adult origin. Their ablation by antibody treatment results in permanent alterations to the adult B-cell repertoire.     

Apparent trans-chromosomal antibody class switch in mice bearing an Igh(a) mu-chain transgene on an Igh(b) genetic background

Native high molecular weight dextran induces a thymus-independent response in BALB/c mice. When the dextran epitope is linked to a protein carrier the response becomes thymus-dependent. IgG antibodies produced after secondary immunization had epitope specificity and idiotope of myeloma M104E. The antibody of M104E (mu, lambda1) is representative for antibodies produced by mice with immunoglobulin haplotype Igh(a) in response to immunization with dextran B1355S. Myeloma product and physiological antibodies share specificity for the alpha(1-3) glucosidic linkage and have idiotopes in common. Mice with haplotypes other than Igh(a) (e.g. Igh(b)) are unable to yield this type of response. A complete rearranged immunoglobulin mu-chain gene with a VDJ-region from BALB/c (Igh(a)) myeloma protein M104E had been introduced into the genome of BALB/c congenic mice having the haplotype Igh(b). As was shown previously in our laboratory the M104E mu-chain transgene confers Igh(a)-type reactivity to Igh(b) mice. In experiments described in this report we used the thymus-dependent form of the antigen to immunize mice bearing the M104E mu-chain, either alone or together with the lambda1-chain, as a transgene on an Igh(b) genetic background. Serological analysis revealed a class switch to IgG very similar to that seen in BALB/c mice with respect to magnitude, kinetics, epitope and idiotope specificity. The pattern of IgG subclass expression was indistinguishable in mu-chain transgenic Igh(b) and normal BALB/c mice. The class switch occurred even though, as is shown here, the transgene had become incorporated in a site not linked to the Igh locus on chromosome 12. We propose a model for this apparent trans-chromosomal class switch recombination which is based on mechanisms known for conventional switch recombination.     

Cell transfer studies in a genetically controlled immune response

Fetal liver cells from 14-17-day old mouse embryos of inbred strains which are genetic responders (C57BL/ 10) or nonresponders (CBA) to a branched synthetic polypeptide [(T, G)-A-L] were injected into reciprocal (nonresponder or responder) strain, lethally irradiated adult mice. These radiation chimeras were then tested for: (a) their ability to respond to (T, G)-A-L; (b) the allotype for specific anti-(T, G)-A-L antibody produced; and (c) the degree of tolerance for CBA (nonresponder) histocompatibility antigens of C57BL/10 (responder) cells taken from high responder chimeras. Eleven high responder radiation chimeras had specific anti-(T, G)-A-L antibody, which appeared to be exclusively responder (C57BL/10) allotype. However, since all the high responder chimeras were predominantly C57BL/10 in total serum immunoglobulin allotype and lymphoid cell type, this result is not conclusive. Six high responder chimeras had C57BL/10 spleen cells which were tolerant of CBA (nonresponder) histocompatibility antigens in the Simonsen discriminant spleen assay.     

Cell autonomous expression of IgD is not essential for the maturation of conventional B cells.

To analyse the function of IgD in vivo, we generated a 'loss of function' mouse model utilizing gene targeting technology. By homologous recombination in a (C57BL/6 x CBA)F1 mouse embryonic stem cell (ES) line one allele of the delta heavy chain gene was rendered non-functional. In chimeric mice obtained after injection of the targeted ES cells into blastocysts derived from severe combined immunodeficient mice we analysed ES cell derived B lymphocytes expressing the targeted or the wild-type allele by using allotype specific reagents. We show that B cells expressing the targeted allele appear in the periphery as IgM+ D- cells at normal frequency. They express the CD23 marker and respond to a T cell dependent antigen. Thus, cell autonomous expression of IgD is neither essential for B cell maturation into an antigen responsive state nor for antigen dependent triggering of the cells into an immune response.     

A Thymus-independent IgG Response against Dextran B512 can be Induced in C57BL but not in CBA Mice, even though both Strains Possess a VHdex Gene

CBA and C57BL mice both possess a VHdex gene coding for antibodies against the alpha 1-6 epitope of dextran B512. After immunization with dextran, CBA mice produce IgM plaque-forming cells (PFC) only, which show regular cyclical fluctuations. The second PFC peak disappeared after injection of dextranase, indicating that it is antigen-dependent. The anti-dextran response in C57BL mice is characterized by only one IgM peak, followed 1 day later by an IgG peak that may exceed the IgM response by a factor of 10. The IgG anti-dextran response in C57BL mice was thymus-independent. CBA mice gave an IgG response to the hapten fluorescein isothiocyanate (FITC) after immunization with FITC-dextran, indicating that dextran can function as a carrier for an IgG response in this strain. Attempts to induce IgG PFC against dextran by immunizing CBA mice with thymus-dependent dextran-protein conjugates consistently failed, althouth the conjugates induced IgG fc in C57BL mice. Spleen cells from CBA mice failed to produce IgG antibodies against dextran after transfer into lethally irradiated C57BL mice, whereas the C57BL spleen cells produced IgG PFC after transfer into CBA mice. The lack of IgG synthesis against the alpha 1-6 epitope of dextran in CBA mice appears to be regulated exclusively at the B cell level and is restricted specifically to the VHdex gene product.     

Quantitative and spectrotypic analysis of paternal IgG2a expression in normal and allotype-suppressed mice.

The synthesis and clonal diversity of IgG2a molecules bearing the paternally inherited immunoglobulin allotype have been examined in the offspring of matings between BALB/c mothers (Igh-1a) and SJL or C57BL/10 males (both Igh-1b) using a sensitive quantitative single radial immunodiffusion in gel assay and isoelectric focusing with autoradiography. In normal litters, the first detectable paternally-marked IgG2a is extensively polyclonal in both F1 crosses (i.e. diversity precedes expression); however, there is a delay of 2-3 weeks in the first appearance of the clonally diverse set of molecules when these are coded by the SJL genome, compared with the C57BL/10. Delayed maturation of allelically-excluded Igh-1b-expressing B cells in the (BALB/c X SJL)F1 may explain the unique susceptibility of these offspring to chronic allotype suppression when exposed to maternal anti-Igh-1b antibodies in early life. We find that, although such suppressed mice may begin life with a (delayed) synthesis of polyclonal IgG2a of paternal allele (Igh-1b), the condition of chronic suppression later imposed in the majority of mice is associated with spectrotype (clonal) simplicity.     

Immunologic induction of reticulum cell sarcoma: donor-type lymphomas in the graft-versus-host model.

The aim of this study was to investigate malignant lymphomas of donor origin induced in F1 mice undergoing a chronic graft-versus-host reaction (GVHR) after injection of parental strain spleen cells. A total of 3 X 10(8) or 4 X 10(8) C57BL/10 spleen cells were administered to 7-8-week-old H-2 incompatible (C57BL/10 X HTG)F1 hybrids either as single or fractionated i.p. injections. Recipients were killed at intervals ranging from 1 month to 1 year after the first injection and their lymphoid cells typed for host-derived and donor-derived histocompatibility antigens. In 49% of the 88 GVH F1 mice, cells failed to be killed by a hyperimmune serum against HTG (H-2g) but reacted normally with antisera to C57BL/10 (H-2b) and, in the 9 cases tested, to theta-C3H. The conclusion that the lymphoid cells of these mice were derived from donor cells was supported by the finding that these animals lacked immunoglobulins bearing host-derived Iga allotype in their serum. Forty-three percent of the GVH F1 mice developed reticulum cell sarcomas (RCS), 32% revealed hyperplastic lymphoreticular tissue, and 25% showed no grossly abnormal changes. Mice with donor-type lymphoid tissues were found in all three groups; 50% of the 38 RCS detected were donor-type neoplasms. The induction of donor-type RCS during the GVHR strengthens the concept of lymphomagenesis through persistent stimulation with antigen(s).     

Defective immunoglobulin M responses to vaccination or infection with Schistosoma mansoni in xid mice.

Mice vaccinated with irradiated Schistosoma mansoni cercariae develop a persistent immunoglobulin M (IgM) antischistosomulum antibody response. To investigate the possible role of antilarval IgM antibodies in the effector mechanism of vaccine-induced immunity, CBA/N mice, which have an X-linked genetic defect resulting in impaired IgM antibody responses to certain antigens, were analyzed for their resistance to a challenge infection. When either infected with unattenuated parasites or vaccinated with irradiated cercariae, mice of this inbred strain failed to produce detectable IgM antibodies to schistosomulum surface membrane and soluble worm antigens. To analyze the effect of this IgM deficiency on immunity, F1 hybrids were constructed between CBA/N females and nondefective C57BL/6J males. As expected, vaccinated (CBA/N X C57BL/6J)F1 females, as well as (CBA/J X C57BL/6J)F1 males and females, produced normal IgM antibodies to both surface antigens and worm antigen extracts. However, such antibodies were not produced by (CBA/N X C57BL/6J)F1 males (hemizygous for xid). Nevertheless, (CBA/N + C57BL/6J)F1 males displayed the same high levels of immunity to challenge infection as (CBA/N X C57BL/6J)F1 females and (CBA/J X C57BL/6J)F1 males and females. These results indicate that vaccine-induced immunity is not dependent on an IgM response to schistosome antigens.     

Induction of persistent autoimmune haemolytic anaemia in mice by combined use of rat erythrocyte preimmunization and chronic GVHR

Female F1 mice (BDF1: C57BL/6 x DBA/2, BCF1: C57BL/6 x C3H/HeN, BBF1: C57BL/6 x BALB/c) were preimmunized with rat erythrocytes and then received splenic T cells from parental female C57BL/6 mice injected with hydrocortisone acetate before killing. In BDF1 and BCF1 mice, direct Coombs' test (DCT) positivity persisted for more than 4 months in almost all mice. They also showed haematological sign of anaemia, and the survival of infused 51Cr-labelled parental C57BL/6 erythrocytes was reduced. In BBF1 mice, DCT positivity returned to negative within 2 months. Autoantibodies were eluted from DCT-positive erythrocytes and their specificities were examined. They were capable of binding equally well to erythrocytes from all strains of mice tested, including the donor mice. They also cross-reacted with rat erythrocytes but not with sheep, rabbit or human erythrocytes. Isotypes of the autoantibodies eluted were also examined. Interestingly, isotypes of the autoantibodies obtained from individual mice were confined to one or other subclass of IgG isotype: IgG1, IgG2a or IgG2b.     

Homogeneous immunoglobulins in the serum of irradiated and bone marrow reconstituted mice: the role of thymus and spleen.

The influence of thymectomy and splenectomy on the frequency and class distribution of homogeneous immunoglobulins (H-Ig) in serum was studied in lethally irradiated (DBA/2 x C57Bl/Rij)F1 mice reconstituted with syngeneic bone marrow. During four follow-up periods in the first 9 months after transplantation, the sham-operated controls and splenectomized animals developed transient H-Ig in an average frequency of 14.2 and 15.7% respectively. There were no marked differences in the incidence of H-Ig within these two groups. In contrast, thymectomized mice and mice both thymectomized and splenectomized showed H-Ig in much higher frequencies (average percentages 31.6 and 36.5, respectively). The highest frequency of H-Ig was observed between 1.5 and 3.5 months after transplantation. H-Ig of the IgG1 and IgG2 subclasses were most frequent in all groups during the first 3.5 months. Later, H-Ig belonging to the IgM class also appeared in somewhat higher numbers. H-Ig of the IgA class was a very rare finding at any time. These results indicate that the presence of the thymus, but not necessarily of the spleen, is an important factor in the regulation of the immunoglobulin heterogeneity during the reconstitution of the immune system in lethally irradiated and bone marrow reconstituted mice.     

Central tolerance induction in natural immunoglobulin-allotype-specific T cells

While self toleance is induced to IgG(b)(2a) in Igh(b / b) mice, an anti-IgG(b)(2a) T cell activity emerges in their Igh(a / a) congenic counterparts. This activity is revealed by postnatal transfer of Igh(a / a) T splenocytes into Igh(a / b) F(1), in which total suppression of IgG(2a)(b) expression is established. Here, we sought to determine whether the natural T cell unresponsiveness to IgG(2a)(b) in Igh(b / b) mice involved a central tolerance. Based on the kinetics of postnatal thymic C(gamma2a)(b) gene expression in Igh(b / b) mice, we transplanted thymi from Igh(b / b) donors of diverse ages into tolerogen-free Igh(a / a) nu / nu recipients. The state of T cell tolerance or responsiveness to IgG(2a)(b) in these reconstituted nu / nu hosts was determined by monitoring the capacity of their splenocytes to induce suppression in Igh(a / b) F(1). These experiments demonstrated that: (i) in the Igh(a / a) nu / nu recipients of adult Igh(b / b) thymi, 33 to 65 % T splenocytes were from nu / nu recipient origin, but these peripheral Igh(a / a) T cells were rendered tolerant to IgG(2a)(b) during their differentiation through the adult Igh(b / b) thymi, (ii) circulating IgG(2a)(b) was not a prerequisite for this tolerance induction, (iii) Igh(b / b) thymic epithelium was unable to induce tolerance to IgG(2a)(b) and (iv) IgG(2a)(b)-producing / presenting cells, colonizing the Igh(b / b) thymi, were certainly responsible of full tolerance induction to IgG(2a)(b).     

Recent insight into class-specific properties of murine immunoglobulins obtained with the help of monoclonal antibodies

BALB/c and C57BL/6 mice were immunized with monoclonal anti-DNP IgE, obtained by fusion of PX63AG8-6-5-3 cells with spleen cells from immunized C57BL/6 or BALB/c mice, respectively. With the antiallotype sera thus produced the allotype of IgE (i.e. 7) could be defined since BALB/c is of the Igh-a and C57BL/6 of the Igh-b allotype. The antiallotype 7b does not cross-react with other allotypes. Antiallotype 7a recognizes in addition to allotype 7a also allotypes 7j, 7d, and 7e as expected. Both the complement fixing and the sensitizing activities of murine anti-TNP and anti-DNP monoclonal antibodies were examined. The minimal amount/ml for complement fixation was 0.46 micrograms from one of the IgG1 antibodies, 0.46 from IgG2a, 12.5 ng from IgM, 0.7 micrograms from IgG3. Complement fixation by IgG1 was unexpected. The minimal amount/ml for mouse PCA was 0.5-0.7 micrograms from IgG1, 1.44-2.3 micrograms from IgG2a, 5.6 micrograms from purified IgG2b and about 15 ng from IgE. Sensitization by IgG2a and IgG2b were new findings. Minimal amount/ml for guinea pig PCA was 0.23 micrograms from IgG2a. Minimal amount/ml for rat PCA was 2 ng from IgE.     

Enhancement in Igha mouse strains of the "natural" suppressive activity of normal T splenocytes against the expression of Igh-1b allotype. I. Molecular aspects of the chronic suppression obtained

Several approaches have been used in our attempts to increase the "natural" ability of normal T splenocytes (Tn) from BALB/c or BC8 mice (both Igha) to induce, in F1 hybrids, a suppression of Igh-1b expression (IgG2a of b haplotype). These heterozygous F1 were produced by mating these Igha mice and their Ighb-congenic partners (CB20 and C57BL/6, respectively). The most powerful approaches were to sensitize the Igha mice by either autologous splenocytes coated with Igh-1b or B splenocytes from Ighb-congenic mice. In F1 having paternally inherited the b haplotype the sensitized T splenocytes (Ts) prepared from such mice are able to induce, like Tn, when injected at birth, a chronic suppression of Igh-1b expression. However, the suppression was established with a much higher efficiency: already at 6 weeks of age in 100% of the F1 treated with 1 x 10(7) Ts, vs. a final rate of 70% progressively reached only at 42 weeks of age in the F1 treated with 4 x 10(7) Tn. In F1 having maternally inherited Ighb the differences were even more pronounced than with 4 x 10(7) Tn, i.e. the suppression induction was almost totally ineffective, whereas with 2 x 10(7)-4 x 10(7) Ts, a rate of 100% treated F1 subjected to suppression was reached at 19 weeks of age. As the productions of IgM, IgD and IgA of the b haplotype were not affected by the suppression, the Ts are believed to act on the Igh-1b+ cells. Attempts were also made to induce allotypic suppression of other b allotypes by the use, as sensitizing cells, of myeloma cells carrying Igh-6b (IgM of b haplotype). We failed in revealing any sign of a T cell reactivity against Igh-6b similar to the reactivity against Igh-1b. The use of Igh-6b+ myeloma cells grown in an Ighb or in an Igha background allowed us to assume that the cells responsible for the sensitization are, in the Ighb B lymphocyte population, either the Igh-1b+ lymphocytes or the lymphocytes having passively adsorbed this allotype, or both.     

Antigen-specific modulation of murine IgE and IgG2a responses with glutaraldehyde-polymerized allergen is independent of MHC haplotype and Igh allotype.

C57BL/6 mice treated with high Mr, glutaraldehyde-polymerized ovalbumin of highly restricted heterogeneity (termed OVA-POL) exhibit IgE responses upon later exposure to unmodified OVA which, at peak, are 1-3% of those observed in untreated controls. Concomitantly, anti-OVA IgG2a responses are elevated 250-1000-fold via an interferon-gamma (IFN-gamma)-dependent mechanism (ref. 4). Here, the impact of OVA-POL treatment on antigen-specific primary and secondary IgE responses is examined in 14 strains of mice. The data indicate that the capacity of this modified allergen to induce pronounced inhibition of IgE responses (75-99%), paralleled by up to 1000-fold increases in IgG2a responses, is not genetically restricted. Moreover, these changes in antibody production were (i) antigen-specific, (ii) isotype-specific and (iii) operated independently of the responder status, MHC or Igh haplotype of the responder mice. In contrast, treatment with unmodified OVA under the same conditions was without effect on IgE production and led to minor increases in anti-OVA IgG2a production.