IgM hybridomas derived from perinatal B cells show a high degree of auto-reactivity and many had demonstrable anti-idiotypic reactivities by binding studies. Selected multispecific antibodies were also shown to have potent idiotype-specific biological activities and if administered at appropriate stages of development could dramatically alter the responses of these mice when challenged with appropriate antigens in adult life. The results obtained suggest that idiotype-directed interactions between neonatal B cells play an important role in the early establishment of the B cell repertoire which is subsequently expressed in adult mice.

A large number of hybridomas were constructed by fusion of B cells from perinatal liver and spleen. Many of these showed multispecificity, high interconnectivity and anti-idiotype (Id) activity. Several of these were subjected to a detailed analysis to evaluate their influence on the developing immune system. A hybridoma BD2 (mu,kappa), derived from 2-day-old liver, was shown to have anti-T15 and anti-J558 activity by inhibition enzyme-linked immunosorbent assay and by in vivo administration. BD2 reduced primary T15 and J558 Id in adult BALB/c by 50%. In contrast, timed administration of this antibody during neonatal periods resulted in enhancement of responses to phosphorylcholine (PC) and alpha (1----3)-linked dextran (Dex) when these mice were challenged as adults. Another hybridoma DB3 (mu,kappa), derived from a lipopolysaccharide-stimulated fetal liver, reacts with GB4-10 (anti-T15) and not with PC. It also reacts with BD2. It is thus anti-anti-Id with respect to T15 and J558. Early administration of this antibody also led to an enhancement of anti-PC and anti-Dex responses, apparently via expansion of a set of intermediate anti-Id BD2-like B cells. In adult mice it suppressed responses to both antigens. A third hybridoma FC4 (mu,kappa), derived from 3-day-old spleen, reacts with GB4-10 as well as EB3-7 (anti-J558). Introduction of this antibody into neonatal mice enhanced anti-Dex responses while in adults it caused suppression of T15 Id. The results presented here suggest a possible role for neonatal anti-Id B cells in the primary activation of antigen-reactive B cells by Id selection.

The isolation of multispecific B cell hybridomas with a variety of anti-idiotype (anti-Id) activities from the lymphoid organs of fetal and neonatal BALB/c mice suggested that the development of the immune system may depend on Id interactions among autologous B cells. In vitro analysis of antibodies secreted by these hybridomas showed extensive sharing of an idiotope defined by the monoclonal antibody FD5-1. Early and timed administration of this antibody during the perinatal period results in a distortion of the phosphorylcholine (PC) and alpha (1----3)dextran (Dex)-specific B cell precursor compartment of the developing repertoire and is reflected by a drastic reduction of antibody responses to these antigens when challenged as adults. These observations provide strong evidence for the involvement of the early appearing multispecific B cells in Id interactions that bring about the uniform development of the normal adult B cell repertoire. Interference with these interactions at critical stages of developmental results in permanent deficiencies in the adult B cell repertoire.

We have examined the immune repertoire and immune response of a mouse that carries transgenes for a mu heavy chain and kappa light chain. The expression of these genes is under the regulation of their own controlling elements. The transgenes are expressed early in ontogeny and are easily detectable from day 13 of gestation onwards. The pre-B cells seem to function normally as they generate IgM-secreting colonies at normal frequencies. Colonies show predominantly the transgenic specificity. Expression of the transgenes is not limited to B cells since around 10%-20% of peripheral T cells and 50% of thymocytes express the mu transgene as an intracellular protein. Ectopic expression of kappa was not seen. The spleen size of the transgenic mouse is decreased by around 20%; this reduction is largely caused by a reduction of the B cell pool. Almost all B cells express the transgenes, only 30% co-express endogenous heavy chain genes and all co-express endogenous light chain genes. Serum Ig levels for IgM and IgA were normal, 20% of the IgM consist of the transgenic product. Serum IgG levels were decreased. T cell functions (helper and cytotoxic) were normal. Immune responses to conventional antigens were impaired, especially in the early phases of the immune response, but after boosting they were virtually normal, except for IgG3 which remained low. Primary antibody responses to T cell-independent antigens of the class II type (bacterially related antigens) were absent, although precursor frequencies for these antigens were within the expected range. The significance of this finding, as it relates to allelic exclusion of Ig genes, is discussed.

focus assays were detected in the spleens of neonatal mice as early as 4 days after birth. The earliest anti-PC B cells were T15-. T15 foci-forming B cells were first detected 6 days after birth and expanded rapidly to constitute greater than 80% of the total PC-specific foci by day 10. Injection of heat-killed S. pneumoniae (R36A) into neonatal mice resulted in priming of the antibody response to PC, with an idiotype profile reflecting that of precursors of foci-forming B cells at the time of antigen administration. Priming of 2-dayold mice with 2 X10 and 2 X10 R36A induced a five-and ten-fold increase in the antibody response to phosphorylcholine 6 to 8 weeks later. However, only 10 to 15% of the serum antibodies expressed the normally dominant T15 idiotype. Doses below 2 xl0 R36A showed no detectable priming activity. PC-specific hybridomas derived from mice injected with 2 X10 R36A 2 days after birth lacked the idiotypic and molecular characteristics typical of T15 antibodies. Antibodies to phosphorylcholine, raised by immunization of 6-week-old mice are normally protective against pneumococcal infection. However, serum antibodies from mice treated with R36A 2 days after birth and responding to phosphorylcholine following challenge with R36A at 6 weeks of age failed to protect against deliberate infection with virulent S. pneumoniae. These observations imply that the antigen phosphorylcholine does not play a role in the selective expansion and dominant expression of the T15 idiotype.