The Casali Lab

During infection, the emergence of neutralizing antibodies highly specific for microbial pathogens, for instance those to human immunodeficiency virus (HIV) type 1 (HIV-1), influenza virus, rabies virus and Salmonella, is crucial for optimal host immune response. An important feature of neutralizing antibodies is that they are heavily mutated (to acquire a high-affinity for microbial antigens), emerging from unmutated V(D)J templates, and class-switched, mostly to IgG. Patients with the hyper-IgM (HIGM) syndrome, resulting from severe impairment in class switching, are prone to viral or bacterial infection, further highlighting the critical role of class switching in effective antibody response.

Research in our laboratory focuses on the mechanisms of immunoglobulin (Ig) somatic hypermutation (SHM) and class switch DNA recombination (CSR). SHM inserts mostly point-mutations in V(D)J region DNA at a high rate, thereby providing a structural basis for the generation of high affinity Ig mutants and their selection by antigen. The SHM machinery preferentially targets the 5'-RGYW-3' (R = A or G, Y = C or T and W = A or T) motif, particularly its 5’-AGCW-3’ (5’-AGCT-3’ and 5’-AGCA-3’) iterations. CSR substitutes an Ig heavy chain (IgH) constant (CH) region, for instance, Cμ, with a downstream CH region, Cγ, Cα or Cε, thereby endowing an antibody with different biological effector functions without changing the structure/specificity of the antigen-binding site (Figure 1).

SHM and CSR are highly regulated processes that unfold mainly in germinal centers (GCs) of secondary lymphoid organs (Figure 2). Antigen-primed B cells are activated in a CD4+ T helper (TH) cell-dependent fashion to undergo SHM and CSR upon engagement of surface CD40 by CD154 on the surface of activated follicular TH (TFH) cells. SHM and CSR can also be induced in a T-independent fashion, driven by other immunological cells that secrete molecules such as BAFF to activate B cells (Figure 3). Exposure to TH cell-secreted cytokines, particularly IL-4 or TGF-β, is critical for CSR to IgG1 and IgE or IgA, respectively. CSR can also be induced in a T-independent fashion, particularly by ligands for toll-like receptors (TLRs). In the presence of B cell receptor (BCR) signaling, ligands for all five major TLRs (TLR1/TLR2 heterodimer and TLR4 on the surface, and TLR7 and TLR9 in the endosome) expressed in mature B cells are capable of inducing CSR as efficiently as CD154 (Figure 4). Finally, we have shown that CD154:CD40-engagement and BCR crosslinking synergize in induction of translesion synthesis (TLS) DNA polymerases, which are critical mediators of SHM, and likely CSR (Figure 5).

Both SHM and CSR require the intervention of activation-induced cytidine deaminase (AID), which is involved in DNA lesion generation, an early step in both processes. We have shown that CD154:CD40-engagement or TLR engagement induces AID, whose expression is further potentiated by BCR crosslinking. More importantly, as we have also shown, in both human and mouse B cells, induction of AID by GC differentiation-inducing stimuli, such as CD154 or LPS and IL-4, requires evolutionarily conserved homeodomain transcription factor HoxC4, which is induced by the same stimuli that also induce AID (Figure 6). We have shown that estrogen recepetors bind to three evolutionarily conserved and cooperative estrogen response elements (EREs) we identified in the HOXC4/HoxC4 promoter to up-regulate HoxC4 expression, thereby inducing AID, CSR and SHM. HoxC4 binds directly to a highly conserved HoxC4/Oct site in the AID gene promoter and activates this promoter in synergy with other transcription factors, such as Oct-1/2, NF-κB and Sp1/Sp3.

AID deaminates deoxycytidines (dCs), particularly those within 5’-WRC-3’ motifs, in single-strand DNA. It also preferentially deaminates dC within 5’-WGCW-3’ in the transcription bubble of S region DNA in CSR. Also, AID is phosphorylated by PKA at Ser38, a prerequisite for AID to bind to replication protein A (RPA) to enhance deamination of transcribed double-strand DNA. The dU:dG mispair resulting from AID deamination can be "replicated over", yielding a dC→dT or dG→dA transition (phase 1a). Alternatively, dU can be deglycosylated by Ung (phase 1b), yielding an abasic site, a DNA lesion that can be bypassed by error-prone TLS polymerases, including pol θ, pol ζ, pol η and Rev1, during DNA replication, or can be processed by the base excision repair (BER) pathway. Alternatively, the dU:dG mispair can recruit the Msh2-Msh6 heterodimer to initiate the MMR cascade (phase 2). Consistent with this two-stage model, Ung-deficeint B cells display not only much reduced CSR (by more than 90%) but also altered spectrum of mutations, i.e., most dC/dG mutations are transitions, around S regions rising during CSR; Msh2-deficient B cells display reduced CSR and much reduced dA/dT mutations, and, finally, B cells doubly deficient in Msh2 and Ung display no CSR and only dC→dT or dG→dA transitions arising from phase 1a (Figure 7). As we have shown, TLS pol ζ and pol θ play important roles in SHM; and Mlh3, an element of the set of mammalian MMR homologs, contributes to phase 2 in both SHM and CSR.

How AID and the CSR machinery target IgH S regions remains to be determined. We have shown that the 5’-AGCT-3’ motif recurs at a high frequency in the core of S regions, particular Sμ, which is used in switching to all isotypes, in the human and mouse (Figure 8). Further, Xenopus laevis Sμ DNA effectively promoted CSR to IgG1 when grafted into the mouse to replace Sγ1 and, conversely, CSR was significantly impaired following deletion of the 5’-AGCT-3’-rich Sμ core. These together with our demonstration that 14-3-3 proteins, a family of seven evolutionarily conserved and highly homologous adaptors, bind to 5’-AGCT-3’ repeats, indicate that 14-3-3 proteins target AID to S regions for CSR to unfold (Movie). Indeed, as we have shown, 14-3-3 proteins are recruited together with AID and PKA-Cα to the upstream and downstream S regions involved in an ongoing CSR event and play an important role in mediating CSR. Further, blocking 14-3-3, deficiency in 14-3-3γ or expression of a dominant negative 14-3-3σ mutant impairs recruitment/stabilization of AID and/or PKA-Cα to S regions and decreases CSR, further confirm the role of 14-3-3 in targeting AID and the CSR machinery.