Preferential assignment of allotype a1 globulin for the production of early IgM anti-para-azobenzenearsonate antibodies in heterozygous a1a3 rabbits.

Rabbits of allotype a1a3 were injected on days 0, 2, and 4 with mixtures containing equal amounts of pigeon erythrocytes (Prbc) coupled to para-azobenzenearsonate (AA) and to para-azobenzene-N-trimethylammonium (TMA). On day 6, the allotypes of antibody from plaque-forming cells (PFC) of the blood were determined by observing the inhibition of plaque formation by anti-allotype sera. Anti-AA PFC appeared to consist for the most part of cells making antibody of allotype a1 since 65% of them were inhibited by anti-a1 serum and only 8% by anti-a3. Anti-TMA PFC, on the other hand, appeared to consist mostly of cells making antibody of allotype a3, since less than 1% of them were inhibited by anti-a1 but 47% by anti-a3. Antibody allotype for spleen PFC was also determined on day 6 and was similar to that found for blood PFC. Anti-AA PFC were inhibited 74% by anti-a1 serum and 15% by anti-a3 whereas anti-TMA PFC were inhibited 19% by anti-a1 and 43% by anti-a3. Serum hemolysin specific for AA hapten from a1a3 animals was also strongly inhibited by anti-a1 serum but not by anti-a3 whereas the converse was true for hemolysin against TMA hapten. The a1a3 rabbits, in whcih the anti-AA was restricted to allotype a1, were mated to produced homozygous a3a3 animals. When the PFC and serum antibodies of these a3a3 offspring were examined by specific inhibition, the anti-AA activity was found to be of allotype a3 rather than being a-negative. The number of anti-AA PFC in the blood of a3a3 rabbits was lower than that in blood of a1a3 or a1a1 animals. In addition, the TMA hapten appeared to inhibit the response to the AA hapten. Thus a1a3 rabbits immunized with AA-Prbc alone had 14-fold more anti-AA PFC or 18-fold higher anti-AA hemolysin titer than a3a3 animals immunized with both AA-Prbc and TMA-Prbc. Our results are discussed in relation to various explanations which have been offered for an imbalance of allotypes in a given antibody.     

Impaired lymphocyte functions in heterozygous rabbits recovering from neonatal allotype suppression

Lymphocytes from heterozygous rabbits suppressed for an allotypic determinant on kappa light chains by exposure to maternally derived antibodies specific for the paternal gene product were analyzed for their capacity to express membrane-bound and secreted immunoglobulin (Ig). Individual cells displaying allotypic membrane Ig (mIg) were enumerated by a rosette test, while Ig-secreting cells were assessed by means of a hemolytic plaque assay. In a group of suppressed rabbits varying in age from 3 to 19 months, the proportion of cells with mIg of the paternal type was markedly higher than that of cells secreting that type of Ig. The same high proportion of lymphocytes displaying mIg of the suppressed type was observed whether lymphocytes from blood, spleen, or lymph nodes of suppressed rabbits were examined. In contrast, similar analyses performed with cells of normal heterozygous rabbits showed no discrepancy between mIg expression and secretion of either allotype. Lymphocytes synthesizing Ig of the paternal type were also defective in responses to lipopolysaccharide (LPS), which stimulates differentiation to Ig secretion in normal B lymphocytes. These results support the idea that B lymphocytes capable of synthesizing the suppressed type of Ig have functional impairments affecting secretion and responses to environmental stimuli.     

In vitro immunoglobulin allotype synthesis by spleen cells of heterozygous rabbits. Specific suppression by anti-allotype antibody

The production of immunoglobulin (Ig) bearing the b4 and b5 allotypic markers by b⁴b⁵ heterozygous spleen cells cultured in vitro was assessed by means of a sensitive and reproducible radioimmunoassay. Ig synthesis was demonstrated by the increasing amounts of the b4 and b5 allotypes appearing with time in the supernatant fluids. To determine the effect of anti-b4 or anti-b5 antibody on the synthesis of the b4 and b5 allotypes, spleen cells from b⁴b⁵ heterozygous rabbits were incubated for 24 hr in the presence of anti-b4 or anti-b5 and then washed and cultured for an additional 4 days. Anti-b4 suppressed the production of the b4 allotype with no effect on b5 production, whereas anti-b5 suppressed the production of b5 allotype with no effect on b4 production. This suppression of allotype synthesis in vitro presumably results from an antigen-antibody reaction occurring on the surface of lymphoid cells by a mechanism which may be similar to that which brings about allotype suppression in vivo for fetal and newborn rabbits.     

Auto-antibody to an Ig VH region allotype: induction of anti-a1 antibody in an a1-suppressed a1a2 heterozygous rabbit.

Two a1a2 heterozygous sibling rabbits were first suppressed for the paternally inherited a1 VH region allotype and then immunized with a1 IgG. Anti-a1 antibody was detected in the serum of one of the rabbits. The anti-a1 auto-antibody reacted with the same amount of a1 IgG as did a conventional anti-a1 allo-antibody. Most of the IgG and IgM of this rabbit was of the a2 allotype and no significant amount of the a1 allotype was detected as would be expected for an a1 suppressed a1a2 heterozygous rabbit. However, allotype suppression in this rabbit is maintained by endogenous anti-allotype antibody. Rabbits with anti-allotype auto-antibody may be exploited to produce litters of heterozygous and homozygous rabbits efficiently suppressed for selected allotypes.     

Production of allotype by bone marrow cells transferred to rabbits subjected to suppressor treatment by allotype

Inbred rabbits of B/J strain were immunized against Salmonella typhi and their bone marrow cells plus LPS were injected into (Chbb: HM X B/J) F1 hybrids at the age of 3-7 weeks, which had received at birth a treatment of the allotype suppression. Antityphoid antibodies bearing the suppressed allotype were found in the serum of the recipients, which showed no signs of the graft versus host reaction.     

Expression of the major cross-reactive idiotype in a primary anti-azobenzenearsonate response

We have studied the occurrence of IgM plaque-forming cells secreting the cross-reactive idiotype (CRI) characteristic of the anti-azobenzenearsonate antibody responses in individual mice of different strains after one injection of the T-independent antigen, p-azobenzenearsonate-Brucella. Under these conditions of stimulation we find that idiotype is not unique to the Igh-1e and Igh-1d allotypes, but is expressed prominently in Igh-1a and Igh-1j strains and to a lesser but significant extent in Igh-1c and Igh-1b strains. We confirmed previous work that idiotype expression in mice hyperimmunized with protein conjugates of azobenzenearsonate is mainly restricted to mice of the Igh-1e allotype, but we find the display to be less marked than in mice given a single injection of the Brucella conjugate. We conclude that the B cell repertoire is inadequately revealed by analysis of serum antibodies of hyperimmune mice. We suggest the apparent correlation of the anti-azobenzenearsonate idiotype to allotype may be influenced by the activity of heavy chain regulatory genes, rather than the presence or absence of structural genes coding for the major CRI. We cannot exclude the possibility that the previously designated CRI-negative strains may express a cross-reactive determinant that is not necessarily a product of the CRI gene family.     

In vitro allotype suppression of spleen cells from immunized rabbits

We tested whether rabbit immune lymphocytes could be suppressed by anti-allotype antibody (Ab) in vitro as shown for normal lymphocytes. Spleen cells (SpC) from rabbits heterozygous at the b locus (b⁴b⁵) of immunoglobulin (Ig) κ chains were treated with IgG preparations of anti-b4 or anti-b5 Ab in vitro for 24 hr (day 1). After this treatment, the SpC were washed and recultured in medium to day 5. The secreted b4- and b5-Ig were quantitated by a radioimmunoassay. SpC from rabbits injected once with sheep red blood cells (SRBC) were allotype-suppressed. Thus, these SpC treated with anti-b4 Ab secreted normal amounts of b5-Ig but secreted much lower amounts of b4-Ig. Similarly, SpC treated with anti-b5 Ab secreted normal amounts of b4-Ig but secreted no detectable b5-Ig. In contrast, SpC from rabbits injected several times with SRBC (hyperimmunized) could not be allotype-suppressed. Hence, the susceptibility of primary immune cells and the resistance of hyperimmune cells to suppression appear to depend on the stage of B-lymphocyte differentiation, presumably because of loss of surface Ig or perhaps because of other changes in the cells as they differentiate during the immune response.     

Preferential expression of VK21E light chains on IdX Ia.7 positive monoclonal anti-I-E antibodies

We previously characterized major (IdX Ia.7) and minor (IdI) idiotopes in a collection of monoclonal alloantibodies reactive with monomorphic (i.e., Ia.7-like) determinants in the structural domain I of the murine class II I-E molecules. In this report, preliminary structural characterization of this antibody family is presented. First, the contribution of isolated H and L chains of the anti-Ia.7 cluster I mAb 41.A to IdX Ia.7 and IdI 41.A idiotope expression was evaluated by testing the capacity of these chains, either isolated or reassociated in homologous or heterologous hybrid Ig, to inhibit the binding of rat or mouse anti-idiotope mAb to IdX Ia.7+ mAb coated plates. It was found that the IdI 41.A idiotope defined by the mouse anti-idiotopic mAb H90-21.1 required the presence of both 41.A H and L chains for complete expression, while the rat mAb-defined IdX Ia.7 idiotope could be detected on isolated and on reassociated 41.A L chain. To evaluate further the structural correlates of the IdX Ia.7 idiotope, H, L, or both H and L chains of 5 A.BY, 4 A.TH and 1 C3H.SW IdX+ anti-Ia.7 mAb, as well as that of 3 A.TH IdX- anti-I-E or anti-I-A and -I-E mAb were subjected to NH2-terminal amino acid sequencing. These analyses demonstrated a) that different H chains corresponding to different subgroups (at least to the VHII and VHIII) could be expressed without apparent modification of IdX Ia.7 idiotope expression and b) that 9 of 11 IdX+ anti-Ia.7 mAb utilized highly homologous L chains of the VK21E subgroup. The relevance of these findings to the genetic control of the idiotypic markers identified in the Ia.7 system is discussed.     

Genetics of a low-density lipoprotein allotype in a cattle.

The paper describes a cattle serum antigen (LdlA1) located on a low-density lipoprotein and detected by single radial diffusion. The specificity is inherited in a simple Mendelian manner and the gene controlling its synthesis is inherited independently from the one controlling the synthesis of the alpha 2 macroglobulin McA1 antigen.     

Effect of VHa locus allotype-suppression on the expression of closely linked VHx, VHy, and Cmun genes in heterozygous rabbits.

Injection of a1x-y-n81/a2x32y33n82 heterozygous rabbit at birth with anti-a2 allotype antiserum suppresses the production of the VHa2 gene product a2 Ig as well as the expression of the closely linked Cmun gene product n82 but not of the closely linked VHx and VHy gene products x32 and x33. This effect is related to the appearance of a2 and n82 on the same IgM molecules whereas a2, x32, and y33 appear on different Ig molecules.