Secondary rearrangements and hypermutation generate sufficient B cell diversity to mount protective antiviral immunoglobulin responses.

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.

Germinal centers without T cells.

Germinal centers are critical for affinity maturation of antibody (Ab) responses. This process allows the production of high-efficiency neutralizing Ab that protects against virus infection and bacterial exotoxins. In germinal centers, responding B cells selectively mutate the genes that encode their receptors for antigen. This process can change Ab affinity and specificity. The mutated cells that produce high-affinity Ab are selected to become Ab-forming or memory B cells, whereas cells that have lost affinity or acquired autoreactivity are eliminated. Normally, T cells are critical for germinal center formation and subsequent B cell selection. Both processes involve engagement of CD40 on B cells by T cells. This report describes how high-affinity B cells can be induced to form large germinal centers in response to (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll in the absence of T cells or signaling through CD40 or CD28. This requires extensive cross-linking of the B cell receptors, and a frequency of antigen-specific B cells of at least 1 in 1,000. These germinal centers abort dramatically at the time when mutated high-affinity B cells are normally selected by T cells. Thus, there is a fail-safe mechanism against autoreactivity, even in the event of thymus-independent germinal center formation.

Mismatch repair co-opted by hypermutation.

Mice homozygous for a disrupted allele of the mismatch repair gene Pms2 have a mutator phenotype. When this allele is crossed into quasi-monoclonal (QM) mice, which have a very limited B cell repertoire, homozygotes have fewer somatic mutations at the immunoglobulin heavy chain and lambda chain loci than do heterozygotes or wild-type QM mice. That is, mismatch repair seems to contribute to somatic hypermutation rather than stifling it. It is suggested that at immunoglobulin loci in hypermutable B cells, mismatched base pairs are "corrected" according to the newly synthesized DNA strand, thereby fixing incipient mutations instead of eliminating them.

Switch in immunoglobulin class production observed in single clones of committed lymphocytes.

Mouse spleen cells, after stimulation with lipopolysaccharide, were cloned in culture. After 4 to 5 days, the daughter cells were stained and examined for immunoglobulin class with double immunofluorescent reagents. A switch of the stained color of these cells was observed, implying a switch from imunoglobulin M to immunoglobulin G production in the progeny of a single B cell.

Lymphocytes of the toad Xenopus laevis have the gene set for promoting tadpole development.

Nuclear transplantation experiments show that differentiated cells, such as lymphocytes, from the adult frog can express the genes necessary for tadpole development. The transplanted cells were proven to be lymphocytes by immunological methods. The origin of the tadpoles that developed after lymphocyte nuclei injections was ascertained by a karyotypic marker.

A quasi-monoclonal mouse.

As a model for studying the generation of antibody diversity, a gene-targeted mouse was produced that is hemizygous for a rearranged V(D)J segment at the immunoglobulin (Ig) heavy chain locus, the other allele being nonfunctional. The mouse also has no functional kappa light chain allele. The heavy chain, when paired with any lambda light chain, is specific for the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). The primary repertoire of this quasi-monoclonal mouse is monospecific, but somatic hypermutation and secondary rearrangements change the specificity of 20 percent of the antigen receptors on B cells. The serum concentrations of the Ig isotypes are similar to those in nontransgenic littermates, but less than half of the serum IgM binds to NP, and none of the other isotypes do. Thus, neither network interactions nor random activation of a small fraction of the B cell population can account for serum Ig concentrations.

Affinity maturation and class switching.

Affinity maturation and class switching of antibodies are temporally, but not mechanistically, related processes. The basis of affinity maturation is the selection, in the germinal centers, of antibodies that bind the antigen better. Early in an immune response, the selection is from the primary repertoire; later, it is from mutants generated by hypermutation at the immunoglobulin loci. Recently, the door has been opened for the study of the molecular mechanism of hypermutation, which is expected to make a major contribution to general biology. Class switching has been studied in the past for its obvious clinical importance, but also at the basic level of DNA recombination. Progress in understanding class switching has been trailing the progress made in V(D)J recombination, but new in vitro systems and gene-targeted mice are closing the gap.

Hypermutation in antibody affinity maturation.

By studying the role of mismatch repair in hypermutation at the immunoglobulin loci, the field of antibody hypermutation has been integrated into the larger area of DNA repair. Trans-acting factors - Ku70, Ku80 and possibly SWAP-70 - have been identified for the temporally related but not mechanistically related immunoglobulin heavy-chain class-switch.

A rapid assay for detecting cellular TdT enzymatic activity.

We have developed a solid-phase assay for the quantification of terminal deoxynucleotidyl transferase (TdT) enzymatic activity in crude cellular extracts. Affinity-purified, polyclonal anti-TdT antibodies are bound to the wells of a microtiter plate, and TdT in extracts is then bound to the immobilized antibodies. The enzymatic activity of the antibody-bound TdT is measured directly in the wells of the microtiter plate. This method yields highly reproducible values, even with samples of low activity. Because it is also technically very simple, it is ideal for determining enzymatic activity for large numbers of clones with limited numbers of cells.

HeLa-LAV, an epithelial cell line stably infected with HIV-1.

An HeLa-LAV cell line was established by infecting and subcloning previously described CD4-expressing HeLa cells with HIV-1. Cells of this line stably synthesize all major HIV proteins, release infectious particles of HIV-1, but do not die even after long term culture. More than 90% of the cells express the envelope protein gp120 on the surface. The cells can be easily and efficiently labeled with 51chromium, and exhibit a low spontaneous release. Because they are susceptible to killing by allogeneic cytotoxic T cells (CTL) when targeted to gp120, they ought to be a useful source of target cells in any kind of HIV-specific killing assays. The cells may also help studies on HIV replication in non-lymphatic/non-monocytic cells. The HeLa-LAV cell line will be freely available from the AIDS Research and Reference Reagent Program.

An immunoglobulin mutator that targets G.C base pairs.

Hypermutation can be defined as an enhancement of the spontaneous mutation rate which the organism uses in certain types of differentiated cells where a high mutation rate is advantageous. At the immunoglobulin loci this process increases the mutation rate > 10(5)-fold over the normal, spontaneous rate. Its proximate cause is called the immunoglobulin mutator system. The most important function of this system is to improve antibody affinity in an ongoing response; it is turned on and off during the differentiation of B lymphocytes. We have established an in vitro system to study hypermutation by transfecting a rearranged mu gene into a cell line in which an immunoglobulin mutator has been demonstrated. A construct containing the mu gene and the 3' kappa enhancer has all the cis-acting elements necessary for hypermutation of the endogenous gene segments encoding the variable region. The activity of the mutator does not seem to depend strongly on the position of the transfected gene in the genome. The mutator is not active in transformed cells of a later differentiation stage. It is also not active on a transfected lacZ gene. These results are consistent with the specificity of the mutator system being maintained and make it possible to delineate cis and trans mutator elements in vitro. Surprisingly, the mutator preferentially targets G-C base pairs. Two hypotheses are discussed: (i) the immunoglobulin mutator system in mammals consists of several mutators, of which the mutator described here is only one; or (ii) the primary specificity of the system is biased toward mutation of G-C base pairs, but this specificity is obscured by antigenic selection.

Inducible expression of the beta 1 subunit of the sodium pump.

The Na,K-ATPase, or sodium pump, a ubiquitous transmembrane enzyme in higher eukaryotes, consists of an alpha and a beta subunit. Here we investigate the expression pattern of the two beta isotypes in mouse B cell lines. Neither primary cells nor cell lines express beta 2. Abelson virus-transformed pre-B cells express beta 1, while B lymphomas and plasmacytomas do not. Thus, beta 1 expression in transformed cells follows that of their untransformed counterparts. Some subclones of pre-B cell line 70Z/3 express beta 1, and others do not, but lipopolysaccharide induces the beta 1-negative cells to become beta 1-positive.

Enhancers of hypermutation.

Hypermutation at the immunoglobulin (Ig) loci increases the mutation rate more than 10(5)-fold over the normal, spontaneous rate. We studied two kinds of cis-acting elements - 3' enhancers and promoters - in a system in which a gene encoding the mu heavy (H) chain (Igm) is transfected in vitro into a cell line with an active Ig mutator. A construct containing a rearranged Igm gene requires the 3' H enhancer for hypermutation at a rate comparable with the one at the endogenous gene segment encoding the H chain variable region (V). Without the 3' enhancer, the basal mutational activity is much lower, but still higher than the normal, spontaneous mutation rate. Replacement of the 3' H enhancer by atopic elements of similar function also supports full hypermutation. Even though these 3' elements are defined as transcriptional enhancers, they do not seem to increase hypermutation via an increase in the rate of transcription. Replacement of the endogenous promoter by the tk promoter slightly increases hypermutability of the construct; thus, no specific sequences in the Ig promoter are likely to target hypermutation.

A theory of allelic and isotypic exclusion for immunoglobulin genes.

Heavy (H) chain binding protein (BiP), which binds to free immunoglobulin H chain of the mu and gamma classes, can be demonstrated in pre-B-cells. It is proposed that the displacement of BiP from H chain by light (L) chain terminates the activity of the enzyme system, L-generase, which catalyzes DNA rearrangement at the L chain loci, generating the complete gene which may or may not be functional. This ensures allelic and isotypic exclusion for the L chain loci. It is further proposed that those cells that productively rearrange both alleles at the H chain locus are eliminated by the "H chain toxicity" effect.

A protein binding specifically to the IgG2b switch region.

The Abelson-virus-transformed mouse pre-B-cell line 18-81 switches almost exclusively from mu to gamma 2b. From nuclear extracts of this cell line, we have isolated a factor that specifically binds to S gamma 2b. After an eight-step purification scheme, in which different types of DNA-affinity chromatography were used as key elements, we obtained a preparation with two narrowly spaced bands at approximately 69 kD on a silver-stained SDS gel. Binding specificity of main-peak fractions of affinity-purified proteins was analyzed by gel shift assays, in which S gamma 2b, but not S mu, competes. The results are consistent with this factor being part of the switch recombinase.

Immunoglobulin mRNA stability varies during B lymphocyte differentiation.

During differentiation of B lymphocytes, the change in the amount of immunoglobulin heavy chain produced is reflected by a change in the steady state level of heavy chain mRNA. At the pre-B cell stage, the earliest stage at which immunoglobulin chain is produced, and later at the small resting B cell stage, there is a low steady state level of heavy chain mRNA. After the small B cell has differentiated to become a plasma cell, the steady state level of heavy chain mRNA is much higher. We confirm that the transcription rate at the immunoglobulin mu heavy chain gene does not change during differentiation from the pre-B cell to the plasma cell stage. In contrast, we show here that differences in the stability of mu mRNA are sufficient to account for the differences in the steady state level at the various differentiation stages.