Kagefumi Todo

Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line.

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2–3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.

B Cell Selection and Affinity Maturation During an Antibody Response in the Mouse with Limited B Cell Diversity

The quasi-monoclonal mouse has limited B cell diversity, whose major (∼80%) B cell Ag receptors are comprised of the knockin VH 17.2.25 (VHT)-encoded H chain and the λ1 or λ2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/λ1 and VHT/λ2 IgM were equally produced, VHT/λ2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded γ-chains could form λ1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/λ1 anti-pNP IgG may not be due to the incompatibility between the γ-chains and the λ1-chain, but may be explained by the fact that VHT/λ1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/λ2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire

Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SWDeltaC, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SWDeltaC cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SWDeltaC cells, although the protein might be highly susceptible to degradation. In DT40-SWDeltaC cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SWDeltaC cells may be useful for constructing Ab libraries for efficient screening of mAbs in vitro.

Conditional transformation of immunoglobulin mutation pattern from gene conversion into point mutation by controlling XRCC3 expression in the DT40 B cell line.

A hypermutating B cell line DT40 is useful for screening antibodies and improving affinity of the selected antibodies in vitro. To perform affinity maturation efficiently, we generated an engineered DT40 line whose immunoglobulin mutation pattern can be transformed from gene conversion into point mutation by conditional suppression of XRCC3 expression.

Enhancement of antibody production from a chicken B cell line DT40 by reducing Pax5 expression

We developed a novel in vitro antibody (Ab) generation system using a hypermutating chicken B cell line (DT40-SW). We suppressed the expression of the Pax5 transcription factor by targeted disruption of the gene to increase Ab production in isolated clones and produce the desired Abs. This single genetic manipulation resulted in a significant enhancement of Ab production without significantly affecting maximum cell density.

IgG1 cytoplasmic tail is essential for cell surface expression in Igβ down-regulated cells.

It has been shown that cytoplasmic tail of the IgG1 B cell receptors (BCRs) are essential for the induction of T-dependent immune responses. Also it has been revealed that unique tyrosine residue in the cytoplasmic tail of IgG2a has the potential of being phosphorylated at tyrosine and that this phosphorylation modulates BCR signaling. However, it still remains unclear whether such phosphorylation of IgG cytoplasmic tail is involved in the regulation of BCR surface expression. In order to approach the issue, we established and analyzed the cell lines which express wild-type or mutated forms of IgG1 BCR. As the result, we found that IgG1 BCR expressed normally on the surface of A20 B cell line independent of the cytoplasmic tail. In contrast, IgG1 BCR whose cytoplasmic tyrosine was replaced with glutamic acid which mimics phosphorylated tyrosine, was expressed most efficiently on the surface of non-B lineage cells and Igβ-down-regulated B cell lines. These results suggest that tyrosine residue in IgG cytoplasmic tail is playing a essential role for the efficient expression of IgG BCR on the cell surface when BCR associated signaling molecules, including Igβ, are down-regulated.

Modulation of Igβ is essential for the B cell selection in germinal center

The positive and negative selection of antigen-reactive B cells take place in the germinal center (GC) during an immune responses. However, the precise molecular mechanisms underlying these selection machineries, including the involvement of antigen receptor signaling molecules, remain to be elucidated. We found that expression levels of Igα and Igβ, which are the essential components of B cell antigen-receptor complex, were differentially regulated in GC B cells and that the expression of Igβ was more prominently down-regulated in a portion of GC B cells. The suppression of Igβ down-regulation reduced the number of GL7+GC B cells and the affinity maturation in T-dependent responses was markedly impaired. In addition, the disease phenotypes in autoimmune-prone mice were ameliorated by blocking of Igβ down-regulation. These results suggest that Igβ down-regulation is involved in the normal positive selection in GC and the accumulation of autoreactive B cells in autoimmune-prone mice.

Erratum: Erratum: Modulation of Igβ is essential for the B cell selection in germinal center

The positive and negative selection of antigen-reactive B cells take place in the germinal center (GC) during an immune responses. However, the precise molecular mechanisms underlying these selection machineries, including the involvement of antigen receptor signaling molecules, remain to be elucidated. We found that expression levels of Igα and Igβ , which are the essential components of B cell antigen-receptor complex, were differentially regulated in GC B cells and that the expression of Igβ was more prominently down-regulated in a portion of GC B cells. The suppression of Igβ down-regulation reduced the number of GL7 + GC B cells and the affinity maturation in T-dependent responses was markedly impaired. In addition, the disease phenotypes in autoimmune-prone mice were ameliorated by blocking of Igβ down-regulation. These results suggest that Igβ down-regulation is involved in the normal positive selection in GC and the accumulation of autoreactive B cells in autoimmune-prone mice. The B cell antigen receptor (BCR) is a protein complex that consists of a membrane-bound immunoglob-ulin (Ig) molecule and the signal transducer, an Igα /Igβ hetero-dimer, and other signaling molecules 1–3. It is well known that the Igα /Igβ hetero-dimer is required for the expression of membrane-bound Igμ chains on the surface of preB cells 1,4,5. Furthermore, Igμ chains expressed on the cell surface of B lineage cells in association with Igα /Igβ hetero-dimer play essential roles in both differentiation and survival of preB cells and mature B cells 6,7. In addition, it has been shown that cell surface expression of the Igα / Igβ hetero-dimer not only supports the expression of cell surface Igμ chains, but also the signal through this complex is further required for the differentiation and survival of B lineage cells 8–10. Hence, it has been widely believed that both Igα and Igβ are expressed in B lineage cells during all maturation stages. After completion of differentiation, mature B cells participate in the humoral immune responses. One of the hallmarks of the humoral immune response is the formation of germinal centers (GCs) following the activation of B cells by an antigen under the influence of T cells 11–13. It is widely known that GC B cells can be classified into two compartments namely centroblasts and centrocytes. Centroblasts are observed in the dark zone and they lack or express only low levels of surface Ig. These cells proceed with somatic hypermutation of their antibody variable genes and proliferate …