Abstract
One of the major mechanisms by which the immune system combats unpredictable foreign pathogens is to produce highly specific and effective antibodies by B cells. Somatic hypermutation (SHM) and class switch recombination (CSR) are two key events required for the production of highly specific and effective antibodies. Both SHM and CSR occur in the germinal center (GC), and are mediated by a common enzyme, activation-induced cytidine deaminase (AID) that specifically mutates the variable regions (IgV) and switch regions (IgS) of immunoglobulin genes by converting cytidines to uridines. It has been shown that in addition to the central deaminase domain, an N-terminal domain is essential to target SHM to the IgV region whereas a C-terminal domain is necessary for Ig CSR. Given its mutagenic nature, it has been postulated that AID may play causative roles in B cell lymphomagenesis. Consistent with this notion, AID is expressed and alternatively spliced in B cell tumors such as B-cell chronic lymphocytic leukemia (B-CLL) and non-Hodgkin’s lymphoma (NHL). However, it remains unclear whether post-GC malignant B cell AID expression and alternative splicing is a consequence of B cell transformation and/or is required to maintain growth and/or malignant B-cell survival, or whether post-GC AID expression and alternative splicing may also occur in normal B cells. To discriminate between these two possibilities, we analyzed AID expression and splicing in normal human B cells. Our data demonstrate that human tonsillar B cells prominently express the full-length AID transcript, and of interest, also express all four alternative transcripts that have been previously described in B-CLL and NHL B cells. To determine if all normal B cells express all of the AID transcripts, we performed limiting dilution of tonsillar B cells and used RT-PCR to detect AID expression. We surprisingly discovered that AID splicing variants are singly expressed by individual B-cells. To address the functional significance of each splicing variant, we considered the possibility that B cells expressing the alternative transcripts may be by-products of the normal GC reaction and are eliminated prior to GC exit. Conversely those B cells may contribute to the memory B cell pool if those AID variants are functional. Therefore, we examined AID expression and splicing in memory B cells using a more sensitive nested PCR approach. Our data show that AID is indeed expressed in peripheral blood memory cells but not in naïve B cells. Moreover, at least one splicing variant was expressed at levels comparable to the full-length AID transcript. Using limiting dilution, we again found that the full-length and alternatively spliced AID transcripts are singly expressed by individual memory B cells. Our results suggest that AID expression beyond the GC stage may play a role in sustaining selection pressure. Since the AID domains specific for SHM and CSR are encoded by different exons, we hypothesize that those variants may function differentially in SHM and CSR and this concept is under current study. Since each B cell only expressed one AID transcript, our data strongly suggest that SHM and CSR are coordinated in a stepwise process by generation of alternative AID splice variants that exhibit selective activity thereby allowing fine-tuning of humoral immunity.
Alternative splicing of RNAs transcribed from the human c-myb gene.
