B cell repertoire in adult antigen‐free and conventional neonatal balb/c mice

In (6)(7)(8). This is not due to biases introduced by transformation or hybridization, since overexpression of the VH7183 genes early in ontogeny was confirmed by "in situ" hybridization methods comparing VH gene family representation in B cells from fetal liver and adult spleen (9) and "natural" plasma cells of neonatal and adult mice (10). A similar preferential usage of the VH7183 gene family occurs in differentiating B cells of the adult bone marrow (11,12). These studies support the notion of developmental control in VH gene family repertoires determined by a nonrandom position-dependent rearrangement preference (13,14). These repertoires evolve thereafter in a strain-specific manner during B-cell maturation (9, 10, 15), but it remains to be established what mechanisms dictate the selection of the VH repertoires expressed in the peripheral B-cell pools of an adult mouse. It is likely that the antigenic composition of the environment may play an important role in the final modulation of the expressed repertoires (10).We have now compared the VH gene family usage in the bone marrow (emergent) and splenic (available and actual) B-cell repertoires of germ-free and conventional (SPF) BALB/c adult mice. We found that VH gene utilization differs in these mice, suggesting that peripheral selection of VH gene repertoires is influenced by environmental antigens.Exogenous antigens could modify the expressed repertoires either by direct stimulation of antigen-specific clones or indirectly by idiotype interactions mediated by the immunoglobulins produced in those responses (16). If this was the case, administration of serum immunoglobulins could lead to changes in the expressed B-cell repertoires. Indeed, we found that the injection of purified isologous serum immunoglobulins into germ-free mice modifies VH gene family utilization. These results suggest that B lymphocytes and their products contribute to the selection of peripheral repertoires.MATERIALS AND METHODS Animals. Inbred BALB/c mice raised in germ-free conditions for 40 generations were from CIBA-Geigy. Control SPF BALB/c mice were from the same stock and breeding facilities or from the Pasteur Institute. As no differences were observed between both stocks of control SPF BALB/c mice, they were used indifferently thereafter. For each experiment, mice were matched for age (8-10 weeks) and sex.Immunoglobulin Treatment. Blood samples from 8-to 9-month-old female BALB/c mice were clotted in the cold and the serum was collected. Protein concentration was adjusted to 10-12 mg/ml with phosphate-buffered saline (PBS), and immunoglobulin was precipitated by addition of saturated ammonium sulfate. The precipitate was sedimented for 60 min at 4000 x g, dissolved in 20 mM Tris HCI/25 mM NaCl, pH 7.9, and dialyzed trice against the same buffer. A DE-52 ion-exchange column was prepared according to Whatman protocol (1 ml of DE-52 per 10 mg of protein). The column was equilibrated with the same buffer, the precipitated dialyzed protein was passed through the column, ...

Section: Discussionmentioning

confidence: 78%

Normal serum immunoglobulins participate in the selection of peripheral B-cell repertoires.

Freitas

Viale

Sundblad

et al. 1991

“…In mice, only a small fraction of this potential repertoire seems to be expressed as functional antibody (2). Utilization of this repertoire appears to be developmentally regulated in a strain-specific fashion (3)(4)(5) with subsequent modification by environmental stimuli (7).…”

mentioning

confidence: 99%

Preferential utilization of conserved immunoglobulin heavy chain variable gene segments during human fetal life.

Schroeder

Wang

1990

233

146

The ability to respond to specific antigens develops in a programmed fashion. Although the antibody repertoire in adults is presumably generated by stochastic combinatorial joining of rearranged heavy variable, diversity, andjoining (VH-DH-JH) and light (VL-JL) chains, experimental evidence in the mouse has shown nonrandom utilization of variable gene segments during ontogeny and in response to specific antigens. In this study, we have performed sequence analysis of 104-day human fetal liver-derived, randomly isolated constant region C, transcripts and demonstrate a consistent preference during fetal life for a small subset of three highly conserved VH3 family gene segments. In addition, the data show that this preferential gene segment utilization extends to the DHQ52 and the JH3 and JH4 loci. Sequence analysis of two "sterile" DH-JH transcripts suggests that transcriptional activation of the JH-proximal DHQ52 element may precede initiation of DH-JH rearrangement and influence fetal Do utilization. Sequence comparisons reveal striking nucleotide polymorphism in allelic gene segments which is poorly reflected in the peptide sequence, implying considerable evolutionary selection pressure. Although vertebrate species utilize a variety of strategies to generate their antibody repertoire, preferential utilization of VH3 elements is consistently found during early development. These data support the hypothesis that VH3 gene segments play an essential role in the development of the immune response.Immunoglobulins are generated by combinatorial joining of rearranged gene segments of the heavy chain variable, diversity, and joining regions (VH, DH, and JH) and light chain regions (VL and JL) (1). Starting with less than 1000 of these germ-line elements, more than 109 different antigen binding sites can be generated even in the absence of eitherjunctional diversity or somatic mutation. In mice, only a small fraction of this potential repertoire seems to be expressed as functional antibody (2). Utilization of this repertoire appears to be developmentally regulated in a strain-specific fashion (3-5) with subsequent modification by environmental stimuli (7).The human neonate is relatively immunodeficient at birth (8). Controlled mobilization of germ-line variable gene segments has been postulated to underlie, in part, the maturation of humoral immunity (5, 9). With these observations in mind, we have concentrated on the use of molecular cloning strategies to dissect the development of the human heavy chain repertoire during fetal life. Because the extent of VH region polymorphism in the outbred human population is undefined, we have chosen to analyze individual fetal samples.Fetal B lymphopoiesis begins in the liver, with pre-B cells first detectable by 8 weeks of gestation (10). We isolated fetal liver mononuclear cells, which are rich in B-cell precursors, and generated oligo(dT)-primed cDNA libraries. We previously reported evidence of preferential usage of VH, DH, and JH gene segments by cloning and sequencing 1...

“…The genes from VH families located at the 3' end of the VH locus are preferentially expressed in pre-B, neonatal, and germ-free adult B-cell repertoires (33,34). A similar bias is also seen in the VH repertoire of autoreactive B cells (35).…”

mentioning

confidence: 88%

Molecular characterization of J558 genes encoding tight-skin mouse autoantibodies: identical heavy-chain variable genes code for antibodies with different specificities.

Kasturi

Yio²,

Bona³

1994

Tight-skin mouse, a mutant strain with a single gene defect, develops cutaneous hyperplasia and specific autoantibodies, like humans affected by scleroderma. The autoantibodies produced in the tight-skin mouse are encoded primarily by heavy-chain variable (Vu) genes from the J558 family. To understand the genetic basis of production of autoantibodies, we have analyzed the structure of J558 genes encoding these autoantibodies. The results showed that J558 genes encoding these antibodies were not derived from a selected germ-line gene(s) or a single subfamily but were derived from genes belonging to diverse J558 subfamilies. However, two prototype VH genes representing two new subfamilies were found to be repeatedly expressed in their germline form in eight independent clones. Autoantibodies with distinct specificities appear to be generated by pairing of similar/identical VH genes with different V,, genes derived from the same or different families. Fourteen of 18 autoantibodies shared a conserved heptapeptide sequence motif, YNEKFKG, in the second complementarity-determining region of heavy chains. Usage of germ-line genes from diverse J558 sub s bearing a common motif to encode autoantibodies suggests a regulatory role for this motif. Thus, selection and expansion of the autoreactive B-cell repertoire in the tight-skin mouse appear to be VH-gene mediated. The frequency of N nucleotide addition at diversity-joining (D-JH) junctions was lower, whereas the frequency of usage of the DFL16 segment was higher. Finally, in contrast to normal and other autoimmune mouse strains, the frequencies of D-D fusions and D inversions were higher in tight-skin mouse total immunoglobulin as well as autoantibody repertoires.Antibody diversity is generated by somatic recombination of diverse germ-line gene segments coding for heavy and light chains of immunoglobulins. In addition, somatic mutation, insertion or deletion of nucleotides at V(D)J junctions, D-D fusion, D inversion, and gene conversion that occur during the recombination process contribute to antibody diversity (1)(2)(3)(4)