Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas

Abstract

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts, is required for this process, but its exact role is unknown. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G*C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.

Forced expression of AID facilitates the isolation of class switch variants from hybridoma cells

“…In two of the AID overexpressing 36-65 subclones (L25A1 and L27A8) the frequency of mutation was much higher (1.7 × 10 −3 and 1.6 × 10 −3 , respectively (Table 1). These V region mutation levels were comparable to those reported earlier in other hybridomas (Martin et al, 2002a). Most of the mutations were transitions and in hotspots and all were in G:C base pairs (Table 1).…”

“…5 × 10 6 cells were transfected with 10 μg full length human AID expressing vector (pCEP4-hAID) or empty vector control linearized with EcoRV and NruI (Martin et al, 2002a), using a GenePulser electroporator (BioRad, Hercules, CA). Cells were distributed in 96-well plates at 10 4 cells/well, selected with hygromycin B (Calbiochem, San Diego, CA) and stable transfectants were detected ~2 weeks later.…”

“…L25 and L27 clones were stably transfected with a vector expressing human AID (hAID) (pCEP4-AID), as well as with the empty vector (pCEP4), as previously described (Martin et al, 2002a). RT-PCR was performed to screen for expression of hAID in the transfectants (data not shown) and only those subclones expressing hAID mRNA were used in subsequent experiments.…”

“…Because expression of AID induces SHM in hybridomas (Martin et al, 2002a), we sequenced the heavy chain V regions of AID transfected 36-65 subclones carried in culture for two months. In control untransfected 36-65 hybridomas after 2 months in culture, the frequency of mutation was ~1 × 10 −4 (Table 1), which is slightly higher than the reported error for the Pfu polymerase (1.3 × 10 −6 mutation frequency/bp/duplication or 4.5×10 −5 for 35 cycles of amplification) (Cline et al, 1996) and in L27 comes from only one mutation.…”

Immunoglobulin V somatic hypermutation in mantle cell lymphoma: mutated genotype correlates with better clinical outcome

“…In this regard, activationinduced cytidine deaminase has been shown to be important in mediating somatic hypermutation. 26,27 In chronic lymphocytic leukemia, there is evidence that cases with high IGH homology express activation-induced cytidine deaminase differently from cases with relatively low homology (o98%), 28 and it has been suggested that this enzyme is defective in a subset of the highly homologous cases and this biochemical defect accounts for their unmutated phenotype. Similar defects may occur in mantle cell lymphoma and these defects may account for the relative lack of V H somatic mutation in a subset of unmutated cases.…”