Ordered expression of various V gene families and acquisition of antigen specificities during ontogeny

In pioneer studies, Silverstein demonstrated that responses of lambs to certain antigens, such as viruses, ferritin, azoproteins, ovalbumin and hemo-cyanin, could be obtained during fetal life, whereas responses to diphtheria toxoid, O antigen, and BCG occurred only after 40 days of postnatal life. Studies on antibody responses and of idiotype expression in young mice have also helped to show that there is a sequential or ordered activation of clones during postnatal life. There are some polysaccharides such as ¡32-6 fructosan, S. tranaroa Iipopolysaccharide (LPS) bearing the a-methyl-D-galactoside immunodominant sugar, or /31-6 galactan, which can elicit an immune response in 1-day-old mice. Immunization with /32-6 fructosan is not paralleled by an increased expression of A48 and UPC10 cross-reactive idiotype (IdX). In contrast, about 25% of LPS-specific antibodies express MOPC387 IdX of a myeloma protein specific for Salmonella tranaroa LPS. Similarly, 1-day-old mice immunized with gum ghatti develop a significant antigalactan plaque-forming cell (PFC) response, of which 30% expressed the X24 idiotype. Studies on the ontogeny of anti-phosphocholine (PC), phenylarsonate (Ars), and tri-nitrophenyl (TNP) antibody responses have shown that the production of antibodies against these antigens, which share the IdX of myeloma proteins with the same specificity, can be induced in 1-week-old mice. Thus the majority of the PC specific precursors bearing T15 IdX can be detected 4-5 days after birth, while cells able to produce IdX4" Ars-specific antibodies are present before day 7 in neonatal mice. Immunization of 1-day-old mice with TNP-LPS and of 1-week-old mice with TNP-Ficoll elicited a significant anti-TNP response. However, the 460Id expressed on the DNP-binding myeloma protein MOPC460 was detected only in 1-week-old mice immunized with TNP-LPS and in 3-week-old mice immunized with TNP-Ficoll. The activation of 460Idf anti-TNP clones in 7-day-old mice coincided with the age when diversification of the anti-TNP response occurs. In the case of al-3 dextran, although the precursors expressing MOPC104Id can be detected during the first week of life, J5581dX precursors appear only 15-22 days after birth and then rapidly become dominant. We have further observed a substantial ontogenic delay in the case of anti-/32-1 fructosan (inulin) responses. A substantial increase in the anti-inulin response bearing IdX has been observed only in 28-day-old mice. This delayed response is associated with a lack of precursors in young animals. A delayed ontogenic response was also observed in the case of al-6 dextran and is also related to the absence of precursors. Fernandez and Moller actually showed that the precursors of anti-a 1-6 dextran can only be detected in 1-month-old mice.

The majority of the data discussed above concern T independent antigens and, therefore, the delayed ontogenic responses are not due to the immaturity of T cells. The sequential activation of some antibody responses cannot be ascribed to a lack of V gene rearrangements in young mice. It seems unlikely that the late maturation of some responses can be explained by the tolerance of the precursors in young animals. In the case of late anti-/32-l fructosan responses, the tolerogen would have to be inulin rather than levan. The lack of responsiveness is limited to j32-l fructosan and not to /32-1 fructosan linkage, and both epitopes are carried by bacterial levan which is an environmental antigen. A more plausible explanation is that the sequential activation is acquired by antigen-independent generative mechanisms or T cell influences. Certain unknown mechanisms may determine the time of particular VH:VK pairing that might be critical for the specificity of the combining sites recognizing particular epitopes.

Finally, the sequential activation of clones expressing a particular set of V genes can be driven by internal forces such as anti-idiotype antibodies. This idea is strongly supported by the data of Vakil and Kearney who, while analyzing the binding specificity for some major IdX of antibodies produced by hybridomas originating from fetal liver or neonates, observed that a high number (7%) of them exhibited anti-Id activity.

There are numerous VH and VK germ-line genes both in mice and humans. Based on protein sequence homology, the VH and VK germ-line genes were classified into several subgroups and now, based on DNA homology, they have been classified into various families. The germ-line genes belonging to a family are grouped in clusters, rarely interspersed, and provide an order on chromosome 12 for heavy chain or on chromosome 6 for VK light chain.

Analysis of V gene family expression in Abelson-transformed pre-B cell lines has shown a preferential usage of 3' families. In fact, VH81X, the most D-proximal member of this family of genes, has been observed in hybridomas of pre-B cell type as well as those prepared from fetal liver. In contrast, different results have been obtained in studies in which the usage of gene family was investigated in nontransfor-

med pre-B cell lines prepared from 6- to 8-week-old nude BALB/c mice. In this study, it was shown that all probes hybridized with detectable intensity to RNA from resting pre-B cells. These data indicate that all VH gene families were transcriptionally active in resting pre-B cells from adult mice. Incubation for 7 days with dendritic cells and mitogen-stimulated T lymphocytes caused a higher expression of the VH7183 family suggesting that the expression of this family is regulated by factors other than ontogeny and, perhaps, is related to specific stages of the differentiation of the B cell lineage. The functional significance of the nonrandom rearrangement of VH genes is of considerable importance in terms of the emerging functional B cell repertoire of the fetus.

Yancopoulos and Alt have hypothesized that the position-dependent preference of Vn family utilization in pre-B cells and neonatal liver is most likely related to a one-dimensional tracking mechanism, mediated by recombinases, during VDJ joining rather than to a dissociative joining related to collisions during three-dimensional diffusion. There are data which indicate that VH genes from one family can be replaced by VH genes from another family in young lymphocytes. It appears that V gene replacement is a rather rare event; however, it may contribute to the establishment of the pre-B cell repertoire. A highly restricted set of VH gene segment usage has also been observed in human fetal B cells. Analysis of human VH repertoire at 130 days of gestation has shown a biased usage of certain JH genes (JH3, JH4 and Jh5) and, also a VH gene designated 56P1 which shows a high homology with murine VH81X, a member of the VH7183 family preferentially rearranged in murine pre-B cells.

Similarly, certain VK families are preferentially expressed during ontogeny. Kaushik and colleagues analyzed the usage of VK families by neonatal murine B cells using an LPS-induced B cell colony assay. The results show that a group of VK families such as VK1, Vk9 and VK8, located in the middle of the VK locus, are highly used by neonatal C57BL/6 mice. Interestingly, the expression of V„21, the most JK proximal family, was not observed among neonatal B cell colonies. These data suggest that VK usage in neonatal mice does not reflect positional bias for the expression of 3' families, rather a preferential usage of VK1 and Vk9 family, located in the center of the VK locus. The differences imply that the mechanisms for VK gene rearrangement and expression differ from those controlling the VH locus.

See also: B lymphocyte differentiation; Bone marrow and hematopoiesis; Bursa of Fabricius; Thymic epithelium: potential role in regulatory T cell tolerance;

Immune response; Thymus; T lymphocyte differentiation.

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