Major histocompatibility complex class II DOA sequences from three Antarctic seal species verify stabilizing selection on the DO locus.

To provide additional support for the sequence conservation and hence the regulatory role of the MHC class II DOA locus, we obtained the nucleotide sequences of exon 2 and exon 3, along with the intervening intron, of the Ross seal, and sequences from the exon 2 region from the Weddell and leopard seals. These are the first reports of the sequences of this locus from a carnivore species. The results demonstrate strong conservation among mammals for the exon sequence and produce a gene genealogy that is consistent in topology with a species tree.

Heavy chain V gene-specific elimination of B cells during the pre-B cell to B cell transition.

As developing B cells acquire their surface Ig (sIg) receptors, they become highly susceptible to sIg-mediated negative selection, a process best exemplified by tolerance induction. Recent studies with sIg transgenic mice have suggested that B cells may become inactivated by tolerogens only after a developmental stage wherein they express low levels of sIgM and during the course of up-regulating their expression of sIgM. To determine whether inactivation of B cells of conventional mice occurs at this or other maturational stages, we have analyzed the ratio of productive vs nonproductive rearrangements of VH81X gene segments in developmental subsets of adult bone marrow cells. Earlier studies had demonstrated that cells whose productively rearranged H chain V region contained a VH81X gene segment were selectively disfavored both during pre-B cell development and subsequent to sIg expression. Contrary to the expectations for elimination by tolerance, no decrease in the proportion of cells expressing productive rearrangements of VH81X was observed as cells matured from the sIgMlow to the sIgMhigh maturational stage. However, a significant decrease in the proportion of productively rearranged VH81X gene segments was observed following the transition from sIg- pre-B cells to sIgMlow immature B cells. Additionally, the proportion of productively rearranged VH81X gene segments was significantly higher in sIgMhigh bone marrow cells than in splenic B cells. These findings demonstrate that B cells are susceptible to H chain-specific elimination at two developmental stages other than that wherein B cells are generally assumed to be negatively selected by tolerance.

Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro.

The increased affinity of memory antibody responses is due largely to the generation and selection of memory B cells that accumulate somatic mutations after initial antigenic stimulation. Further affinity maturation and mutation also accompany subsequent immunizations. Previous studies have suggested that, like primary antibody-forming cell (AFC) clones, secondary AFC do not accumulate further mutations and, therefore, the origins of progressive affinity maturation remain controversial. Here, we report the generation of somatically mutated memory B cell clones in vitro. Our findings confirm the existence of a naive B cell subset whose progeny, rather than generating AFC, somatically mutate and respond to subsequent antigenic stimulation. Interestingly, upon stimulation, a subset of memory B cells also generates antigen-responsive cells that accumulate further somatic mutations.

Specific immunoglobulin cDNA clones produced from hybridoma cell lines and murine spleen fragment cultures by 3SR amplification.

The isothermal 3SR amplification method has been employed to assist in cloning the VL and VH genes from cells of hybridomas and splenic fragment cultures expressing antibodies for phosphorylcholine (PC) and estradiol (E2), respectively. As a first step, pools of degenerate primer pairs were identified complementary to immunoglobulin light and heavy chain variable (V) genes and capable of amplifying immunoglobulin RNA specifically at 42 degrees C. To evaluate the functionality of the 3SR-cloned immunoglobulin genes, anti-PC VH and VL cDNAs were joined together to form a single chain (sc) antibody construct and were expressed in Escherichia coli under the regulation of the alkaline phosphatase (phoA) promoter. Similarly, the combination of a murine spleen fragment and 3SR methodologies were employed to clone a selected pool of cDNAs for cultures producing anti-estradiol antibodies. This approach of using the murine spleen fragment and 3SR isothermal amplification offers the advantages of B-cell follicle architecture for antigen-driven B-cell maturation and proliferation and RNA-specific amplification, respectively. The potential utility of these advantages for the production of monoclonal antibodies and for providing the capability of studying memory B-cell development are discussed.

The use of Bayesian analysis of PCR-derived genomic DNA sequences to enable a biologically relevant interpretation of immunoglobulin variable region gene expression.

We analyzed by means of polymerase chain reactions (PCRs) and DNA sequencing techniques the immunoglobulin heavy chain variable region genes of bone marrow B lineage cells. We first formulated an explanatory model to guide understanding of the biological mechanisms determining both the size of the total available pool of relevant genes and clonal expansion following heavy chain gene rearrangement. We then followed Box's paradigm of criticism and estimation to interpret our experimental findings.

Interrelating B cell subpopulations and environmental regulation with the expression of three tiers of repertoire diversity.

The B cell repertoire consists of three tiers of clonotype diversity. One tier, which is the product of H chain V region rearrangements in the absence of N additions, is of limited diversity (less than 10(8) clonotypes) so that clonotypes of this tier would be expected to recur within and among B cells of individuals of an inbred strain. These clonotypes, therefore, could be subjected to, and conserved by, evolutionary selective pressures such as those imposed by ubiquitous bacterial pathogens. The second tier of clonotypes is created by H chain V region rearrangements that include N additions, and is, therefore, exceedingly diverse. Clonotypes of this tier would be unlikely to recur; however, by providing maximal diversity they would ensure protection against a wide spectrum of pathogens. The third tier of diversity is that which is generated by the superimposition of somatic mutations on clonotypes of the other two tiers. This tier of clonotypes is reflective of the refinement of specificities that are destined for expression in memory B cells. B cells exists as three distinct subpopulations, Ly-1 B cells, conventional primary B cells and memory B cells. These subpopulations differ functionally, developmentally, and by the extent to which they are impacted by immunoregulatory processes. Furthermore, B cells of these subpopulations differentially express the three tiers of clonotype diversity.

Predominance of nonproductive rearrangements of VH81X gene segments evidences a dependence of B cell clonal maturation on the structure of nascent H chains.

Ig H chain V regions using the VH81X gene segment were PCR amplified from genomic DNA obtained from either splenic B cells or surface (s)Ig- bone marrow cells of BALB/c mice. Sequence analysis demonstrated that 93% of VH81X containing H chain V region genes in splenic B cells were rearranged nonproductively. Furthermore, 74% of rearrangements of VH81X among sIg- bone marrow cells were nonproductive. This contrasts with previous results obtained for rearrangements of members of the VH36-60 gene segment family among sIg- cells wherein, as a consequence of extensive clonal expansion after productive H chain V gene rearrangement, 80% of rearrangements were productive. The low proportion of productive rearrangements of VH81X is interpreted as indicating that most productive rearrangements of VH81X cannot facilitate clonal expansion, which would support the hypothesis that selection for clonal expansion and maturation is dependent on the amino acid sequence of nascent H chains. Additionally, because most productive rearrangements of VH81X cannot facilitate clonal maturation but do appear to mediate allelic exclusion, these processes are likely to be regulated independently.

The expression of the Ig H chain repertoire in developing bone marrow B lineage cells.

Analyses of rearranged Ig H chain V region genes of bone marrow pre-B cells demonstrate extensive sequence diversity, particularly within the third hypervariable region (HCDR3). This diversity is constrained, however, through preferential utilization of certain D gene segments and possibly VH gene segments and a preponderance of productive rearrangements, primarily those expressing D gene segments in a preferred reading frame. The predominance of productively rearranged V genes with D regions translated in a preferred frame, is, at least in part, the consequence of selective clonal expansion encompassing at least five to six divisions subsequent to VH-D-JH rearrangement. Selection for clonal expansion appears to be dependent on recognition of the nascent H chain product of certain productively rearranged genes.

Primary antibody-forming cells and secondary B cells are generated from separate precursor cell subpopulations.

Two precursor cell subpopulations have been isolated from the spleen cells of nonimmune mice. The major B cell subpopulation binds high levels of the J11D monoclonal antibody and, upon T cell-dependent antigenic stimulation, gives rise to primary antibody-forming cell clones but not secondary B cells. A minority of the 10%-14% of Ia+ precursors that bind low levels of J11D (J11Dlo) also generate antibody-forming cell clones after primary stimulation. However, over 70% of J11Dlo precursors yield no primary antibody-forming cell clones but instead give rise to secondarily responsive B cells. The existence of a distinct precursor cell subpopulation that is responsible for the generation of B cell memory is further evidenced by the distribution of variable region clonotypes among J11Dlo primary precursors, which resembles the clonotype patterns of secondary B cells, and by the accumulation of somatic mutations in their clonal progeny.