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Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA(3-286)) from strain R36A. Abs to PspA(3-286) were encoded by diverse V(H) and V(kappa) families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 +/- 0.5) and 8-9 (8.7 +/- 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D(H) gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D(H) gene-derived amino acids did not prevent anti-PspA(3-286) mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA(3-286) polyclonal Ab response in PspA(3-286)- and R36A-immunized mice. Compared with PspA(3-286)-immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D(H) genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D(H)-less Abs.

Lineage trees of mutated rearranged Ig V region sequences in B lymphocyte clones often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation. In this study, we use a novel method for analyzing lineage tree shapes, using terms from graph theory to quantify the differences between primary and secondary diversification in rabbits and chickens. In these species, Ig gene diversification starts with rearrangement of a single (in chicken) or a few (in rabbit) V(H) genes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and to secondary diversification during immune responses in germinal centers (GCs). We find that, at least in rabbits, primary diversification appears to occur at a constant rate in the appendix, and the type of Ag-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut-associated lymphoid tissues may play in early development of mammalian immune repertoires. Additionally, the data indicate a higher rate of hypermutation in rabbit and chicken GCs, such that the balance between hypermutation and selection tends more toward mutation and less toward selection in rabbit and chicken compared with murine GCs.

The young rabbit appendix is a dynamic site for primary B cell repertoire development. To study diversification patterns during clonal expansion, we collected single appendix B cells from 3- to 9-wk-old rabbits and sequenced rearranged H and L chain genes. Single cells obtained by hydraulic micromanipulation or laser capture microdissection were lysed, PCR amplified, and products directly sequenced. Gene conversion-like changes occurred in rearranged H and L chain sequences by 3-4 wk of age. Somatic mutations were found in the D regions that lack known conversion donors and probably also occurred in the V genes. A few small sets of clonally related appendix B cells were found at 3-5 wk; by 5.5 wk, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found previously in splenic germinal centers where an immunizing Ag was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3, whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign Ags. This diversity of combining sites within B cell clones supports the proposed role of appendix in generating the preimmune repertoire.

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.

We used PCR to amplify rearranged VHDJH genes in single cells collected by micromanipulation from splenic germinal centers of immunized adult rabbits. In the course of the study, the objective of which was to analyze diversification of rearranged VHDJH sequences, we were surprised to find cells 7 and 10 days after immunization with rearranged VH1a2 as well as a-negative (y33 and x32) sequences that were identical or close to germline (10 or fewer changes). About 58% (82/140) of the sequences had unique CDR3 regions and were unrelated. In seven different germinal centers, we found one to four different clones with two to seven members. Clonally related cells underwent diversification by hypermutation and gene conversion. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. The attractive idea that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life, is challenged by the current finding. Although a major population of B lymphocytes may be generated early in life, diversified extensively, and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. Although considerable repertoire diversity is generated in young rabbits, mechanisms for continued generation of B cell receptor diversity are retained in adult life, where they may confer survival advantage.

We investigated the molecular basis for the appearance of V(H)a2 allotype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of V(H) genes because of a small deletion encompassing V(H)1a2, the 3'-most gene in the V(H) locus. The V(H)1 gene is the major source of V(H)a allotype because this gene is preferentially rearranged in normal rabbits. In young homozygous ali/ali animals, the levels of a2 molecules found in the serum increase with age. In adult ali/ali rabbits, 20 to 50% of serum Igs and B cells bear a2 allotypic determinants. Previous studies suggested that positive selection results in expansion of a2 allotype-bearing B cells in the appendix of young mutant ali/ali rabbits. We separated appendix cells from a 6-wk-old Alicia rabbit by FACS based on the expression of surface IgM and a2 allotype. The VDJ portion of the expressed Ig mRNA was amplified from the IgM+ a2+ and IgM+ a2- populations by reverse transcriptase-PCR. The cDNAs from both populations were cloned and sequenced. Analysis of these sequences suggested that, in a2+ B cells, the first D proximal functional gene in Alicia rabbits, V(H)4a2, rearranged and was altered further by a gene conversion-like mechanism. Upstream V(H) genes were identified as potential gene sequence donors; V(H)9 was found to be the most frequently used gene donor. Among the a2- B cells, y33 was the most frequently rearranged gene.