Overview

Our lab seeks to understand the processes that promote antibody diversification. We use molecular, biochemical, cellular, genetic, and immunological approaches to understand how B cells modify the DNA coding sequence of the antibody. Although the primary function of antibody diversification is to mount the most effective immune response, aberrant DNA modifications can activate genes that can cause cancer. These studies are critical in understanding how we can manipulate antibody diversity to drive more effective immune responses and how specific proteins are redirected towards permitting cancerous cell growth rather than immunity.

Immunity and Immunoglobulins

Mammals have an innate and adaptive immune system that protects them from pathogenic viruses and bacteria. Cells from the innate immune system, such as macrophages and natural killer cells, respond immediately to eliminate the virus or bacteria because these cells recognize foreign molecules non-specifically. Cells of the adaptive immune system (T and B cells) respond more slowly but specifically. T and B cells recognize a unique molecule (or antigen) on the virus and bacteria. The development of antigen-specific T and B cells is genetically programmed through a DNA recombination process called V(D)J recombination, which allows T cells to express antigen-specific receptors (TCRs, T cell receptors) and B cells to produce plasma membrane-bound immunoglobulins (antibodies). Antigen-activated B cells can develop into immunoglobulin secreting cells and further alter the immunoglobulin coding genes through class switch recombination (CSR) and somatic hypermutation (SHM).

CSR alters the isotype of the expressed immunoglobulin through a DNA double‐strand break and non‐homologous end joining (NHEJ) recombination reaction, whereas SHM generates untemplated mutations in the antigen binding domain of the immunoglobulin, which is commonly referred to as the variable region. Both CSR and SHM require the activity of activation‐induced cytidine deaminase (AID), a single‐stranded DNA (ssDNA) cytidine deaminase. AID‐deficiency leads to a complete block in CSR and SHM in humans and mice, while promiscuous AID activity generates mutations and translocations in non-immunoglobulin genes.

To minimize damage at non-immunoglobulin genes, AID is regulated transcriptionally and post‐transcriptionally. AID is phosphorylated on Ser38 (pS38‐AID) by PKA (cAMP‐dependent protein kinase A) at recombining switch (S) regions (Vuong et al. 2009). Mice genetically engineered to express AID(S38A) have a significant block in CSR and SHM (Cheng et al. 2009). Our published data (Vuong et al. 2013) showed that pS38-AID interacts with APE1 (apurinic/apyrimidinic endonuclease 1) to generate DNA breaks in S regions. Interestingly, the phosphorylation of AID is dependent on the formation of DNA breaks and the DNA repair kinase ATM (ataxia telangiectasia mutated). These data suggest a positive feedback loop whereby AID-induced deamination of ssDNA at recombining S regions activates an ATM- and PKA-dependent pathway for AID phosphorylation, which in turn recruits APE1 to the S regions to generate DNA breaks. Thus, a DNA repair pathway involving ATM is required for programmed DNA breaks during CSR.