Gareth T. Williams

Immunoglobulin Isotype Switching Is Inhibited and Somatic Hypermutation Perturbed in UNG-Deficient Mice

BACKGROUND We have previously proposed that deamination of cytosine to uracil at sites within the immunoglobulin loci by activation-induced deaminase (AID) triggers antibody diversification. The pattern of diversification (phase 1 or 2 hypermutation, gene conversion, or switch recombination) is viewed as depending on the mode of resolution of the dU/dG lesion. A major resolution mode involves excising the uracil, an activity that at least four different enzymes can accomplish in the mouse. RESULTS Deficiency in UNG uracil-DNA glycosylase alone is sufficient to distort the pathway of hypermutation in mice. In ung(-/-) animals, mutations at dC/dG pairs are dramatically shifted toward transitions (95%), indicating that the generation of abasic sites (which can induce transversions) has been inhibited. The pattern of substitutions at dA/dT pairs is unaffected. Class-switch recombination is substantially, but not totally, inhibited. CONCLUSIONS The results provide strong support for the DNA deamination model for antibody diversification with respect to class-switching as well as hypermutation and, in the context of this model, suggest that (i) UNG is the major mouse DNA glycosylase responsible for processing the programmed dU/dG lesions within the immunoglobulin locus; (ii) the second (dA/dT-biased) phase of mutation is probably triggered by recognition of the initiating dU/dG lesion; and (iii) switch recombination largely proceeds via formation of an abasic site, although (iv) an UNG-independent pathway of switch recombination exists, which could reflect action by another uracil-DNA glycosylase but might alternatively be explained by a distinct pathway of resolution, for example, one involving MSH2/MSH6 recognition of the dU/dG lesion.

Hyperresponsive B cells in CD22-deficient mice.

CD22 is a surface glycoprotein of B lymphocytes that is rapidly phosphorylated on cytoplasmic tyrosines after antigen receptor cross-linking. Splenic B cells from mice with a disrupted CD22 gene were found to be hyperresponsive to receptor signaling: Heightened calcium fluxes and cell proliferation were obtained at lower ligand concentrations. The mice gave an augmented immune response, had an expanded peritoneal B-1 cell population, and contained increased serum titers of autoantibody. Thus, CD22 is a negative regulator of antigen receptor signaling whose onset of expression at the mature B cell stage may serve to raise the antigen concentration threshold required for B cell triggering.

Developmental regulation of IgM secretion: The role of the carboxy-terminal cysteine

B lymphocytes do not secrete IgM, and plasma cells only secrete IgM polymers. Here we show that both events are attributable to the tailpiece found at the carboxyl terminus of mus chains, and we specifically implicate Cys-575. Thus, if Cys-575 was mutated, IgM was secreted by B cells. Similarly, a mutant IgG containing a mus tailpiece became largely retained within the cell; secretion was restored upon mutation of the tailpiece cysteine. Removal of Cys-575 also allowed hypersecretion of monomeric IgM by plasmacytoma cells. Following further removal of Cmu1, heavy chains were secreted in the absence of light chains. Thus, in B and plasma cells, Cys-575 is involved both in the polymerization of IgM and in intracellular retention of unpolymerized intermediates.

Somatic hypermutation at A·T pairs: polymerase error versus dUTP incorporation

Somatic hypermutation of immunoglobulin genes occurs at both C·G pairs and A·T pairs. Mutations at C·G pairs are created by activation-induced deaminase (AID)-catalysed deamination of C residues to U residues. Mutations at A·T pairs are probably produced during patch repair of the AID-generated U·G lesion, but they occur through an unknown mechanism. Here, we compare the popular suggestion of nucleotide mispairing through polymerase error with an alternative possibility, mutation through incorporation of dUTP (or another non-canonical nucleotide).

Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers

The V regions of immunoglobulin κ transgenes are targets for hypermutation in germinal centre B cells. We show by use of modified transgenes that the recruitment of hypermutation is substantially impaired by deletion of the nuclear matrix attachment region (MAR) which flanks the intron‐enhancer (Ei). Decreased mutation is also obtained if Ei, the core region of the κ3′‐enhancer (E3′) or the E3′‐flank are removed individually. A broad correlation between expression and mutation is indicated not only by the fact that the deletions affecting mutation also give reduced transgene expression, but especially by the finding that, within a single mouse, transgene mutation was considerably reduced in germinal centre B cells that poorly expressed the transgene as compared with strongly expressing cells. We also observed that the diminished mutation in transgenes carrying regulatory element deletions was manifested by an increased proportion of B cells in which the transgene had not been targeted at all for mutation rather than in the extent of mutation accumulation once targeted. Since mutations appear to be incorporated stepwise, the results point to a connection between transcription initiation and the clonal recruitment of hypermutation, with hypermutation being more fastidious than transcription in requiring the presence of a full complement of regulatory elements.

Mice carrying a CD20 gene disruption

Abstract CD20 is a hallmark antigen of B lymphocytes. Its expression is restricted to precursor and mature B cells but it is not expressed on plasma cells. The protein is a membrane-embedded phosphoprotein that appears likely to transverse the membrane four times. Its function is unknown although CD20 has been variously proposed to play a role in B-cell activation, proliferation, and calcium transport. A unique homologue of human CD20 has been described in mouse, which also shows a B-cell-specific pattern of expression. Here we describe the generating of mice carrying a CD20 gene disruption. So far, we have failed to detect any major effect of the gene disruption on the differentiation and function of B lymphocytes as judged by the expression of surface markers, antigen receptor signaling, proliferative responses, or calcium uptake. We did note, however, that the mice homozygous for the gene disruption [generated by intercrossing (129 × C57BL/6)F1CD20+/- heterozygotes] showed a substantial depletion of the sub-population of peritoneal B cells that lack expression of the B220 (RA3–6B2) isoform of CD45. The loss of the IgM+ 6B2- peritoneal B cells is not, however, attributable to the CD20 gene disruption itself. Rather, it segregates with a polymorphic difference between the 129 and C57BL/6 strains that is linked to the CD20 locus which, intriguingly, is itself close to the CD5 gene. This demonstrates that caution must be exercised when comparing the phenotypes of F2 litter-mates generated from crosses between 129 embryonic stem-cell-derived chimeras and mice of other strains.

Acceleration of intracellular targeting of antigen by the B-cell antigen receptor: importance depends on the nature of the antigen–antibody interaction

The B‐cell antigen receptor (BCR) internalizes bound antigen such that antigen‐derived peptides become associated with emigrating major histocompatibility complex (MHC) class II molecules for presentation to T cells. Experiments with B‐cell transfectants reveal that BCR confers a specificity of intracellular targeting since chimeric antigen receptors which internalize antigen by virtue of a heterologous cytoplasmic domain do not necessarily give rise to presentation. In contrast, however, previous studies have shown that antigen binding to irrelevant cell surface molecules (e.g. transferrin receptor, MHC class I) can ultimately lead to presentation. The solution to this paradox appears to be that the intracellular targeting by BCR actually reflects an acceleration of antigen delivery. Depending on the nature of the BCR–antigen interaction, this accelerated targeting can be essential in determining whether or not internalization leads to significant presentation. Physiologically, the accelerated delivery of antigen by BCR could prove of particular importance early in the immune response when antigen–BCR interaction is likely to be poor.

Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines

We show that iterative antigen-mediated selection of B-cell lines that constitutively hypermutate their immunoglobulin V genes during culture can be exploited to generate antibodies in vitro. From Ramos, a hypermutating human B-cell line expressing IgM of unknown specificity, we derived descendants that exhibit stepwise improved binding to streptavidin. Binding is initially conferred by mutations in complementarity-determining regions (CDRs), but maturation is due to strategic framework mutations. A more powerful system is provided by a hypermutating chicken B-lymphoma line, owing to its rapid proliferation, high rate of mutation accumulation, and genetic tractability. Starting from a single cell, we selected parallel lineages of derivatives, making mutated antibodies of increasing affinity to independent test antigens. Selection is initiated at an exceedingly low affinity threshold, but antibodies can be delivered with nanomolar affinities. The strategy could prove useful for in vitro generation of antigen-specific monoclonal antibodies and may be extendable to the maturation of other protein–ligand interactions.

Production of antibody-tagged enzymes by myeloma cells: Application to DNA polymerase I Klenow fragment

Myeloma DNA expression systems can be used for the synthesis and secretion of antibody/enzyme recombinant molecules. Here we describe the construction of a myeloma cell-line that secretes a hapten-specific antibody/enzyme hybrid molecule, in which the antibody Fc portion has been replaced by the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). This Fab-PolIk hybrid molecule is secreted in good yield from the myeloma transfectants, can be purified to homogeneity in a single step on hapten-Sepharose columns, and exhibits PolIk activity as judged by its use in dideoxy nucleotide sequencing. Thus Fab-PolIk can be used for the same purposes as conventional PolIk but has the advantage that it is easily purified to homogeneity in a one-step purification from culture medium.

Dependence of antibody gene diversification on uracil excision

Activation-induced deaminase (AID) catalyses deamination of deoxycytidine to deoxyuridine within immunoglobulin loci, triggering pathways of antibody diversification that are largely dependent on uracil-DNA glycosylase (uracil-N-glycolase [UNG]). Surprisingly efficient class switch recombination is restored to ung−/− B cells through retroviral delivery of active-site mutants of UNG, stimulating discussion about the need for UNGs uracil-excision activity. In this study, however, we find that even with the overexpression achieved through retroviral delivery, switching is only mediated by UNG mutants that retain detectable excision activity, with this switching being especially dependent on MSH2. In contrast to their potentiation of switching, low-activity UNGs are relatively ineffective in restoring transversion mutations at C:G pairs during hypermutation, or in restoring gene conversion in stably transfected DT40 cells. The results indicate that UNG does, indeed, act through uracil excision, but suggest that, in the presence of MSH2, efficient switch recombination requires base excision at only a small proportion of the AID-generated uracils in the S region. Interestingly, enforced expression of thymine-DNA glycosylase (which can excise U from U:G mispairs) does not (unlike enforced UNG or SMUG1 expression) potentiate efficient switching, which is consistent with a need either for specific recruitment of the uracil-excision enzyme or for it to be active on single-stranded DNA.