Research in our laboratory focuses on the mechanisms underlying maturation of antibody and autoantibody responses, with emphasis on somatic hypermutation (SHM) and class switch DNA recombination (CSR) of the immunoglobulin (Ig) genes in B lymphocytes. We aim to identify novel elements that regulate these processes, define biochemical properties and functions of those elements, characterize their physiological and/or pathological roles and, eventually, manipulate immunity and control autoimmunity, allergy, inflammation and cancer.
Antibodies (immunoglobulins) are central mediators of immunity (Casali, Lewin's Genes XI, 2013). They directly neutralize microbial pathogens, such as human influenza virus, as well as pathogen products; they can also recruit molecular and cellular immune effectors to eradicate infections and tumor cells. A key feature of neutralizing antibodies is that they are heavily mutated in their V(D)J regions through SHM, which inserts mostly point-mutations at a high rate to generate high affinity Ig mutants, and class-switched, e.g., IgG, IgA or IgE, through CSR, which replaces the expressed CH exon cluster in the Ig heavy chain (IgH) locus, for example, Cμ for IgM, with Cγ, Cα or Cε
Class-switched antibodies possess different biological effector functions: IgG1 and IgG3 (in human) are effective against viruses, IgG2 against encapsulated bacteria, IgG4 and IgE against large extracellular parasites, and IgA1 and IgA2 against pathogenic bacteria at the mucosae. Aberrant SHM and CSR result in diseases ranging from immune deficiencies, systemic or organ-specific autoimmunity, atopic IgE reactions (e.g., those in asthma and anaphylaxis) to neoplastic transformation.
SHM and CSR unfold mainly in germinal centers (GCs)
, which develop in response to foreign antigens in secondary lymphoid organs in a manner dependent on follicular T helper (TH) cells and expression of Drak2 (Al-Qhatani et al., Autoimmunity 41: 341-352, 2008). In GCs, B cells undergo CSR upon engagement of surface CD40 by CD154 on the surface of activated TFH cells. CSR can also be induced by T-independent primary stimuli outside GCs. These stimuli, as recently shown by us (Pone et al., Nature Commun. 3: 767, 2012), include dual engagement of B cell receptor (BCR) and Toll-like receptors (TLRs), such as TLR1/2, TLR4, TLR7 and TLR9
TLRsare a family of conserved pattern recognition receptors that recognize microbe-associated molecular patterns; they are highly expressed in B cells as well as innate immune cells. TLRs can also boost the T-dependent antibody response in a manner dependent on B cells. The enhancement effect of TLRs is emphasized by the existence of TLR agonists in several new-generation vaccine adjuvants (Pone et al., Front. Biosci. 17: 2594-2615, 2012). TLR activation can prime antigen-specific B cells to express high levels of GC cell markers, and initiate and sustain the GC reaction. Dual BCR and TLR engagement also generates class-switched B cells, such as Igγ+ B cells, which can respond to antigen faster than naive Igμ+ or Igδ+ B cells (Figure 4).
T-dependent and T-independent primary CSR-inducing stimuli require the cooperation of secondary stimuli for switching to predetermined Ig isotopes, e.g., IL-4 for CSR to IgG1 and IgE, TGF-β for CSR to IgA or IFN-γ for CSR to IgG2a. These cytokines thus adapt antibody effector functions to different milieus, such as IgA in the mucosae, where TGF-β is produced at high levels. The synergistic effect of primary and secondary stimuli is mediated by the combinatorial interplay of their respective downstream transcription factors for the induction of chromatin accessibility in the upstream (donor) and downstream (acceptor) switch (S) regions that will be recombined (Xu et al., Nature Rev. Immunol. 12: 517-531, 2012). These S regions also undergo active transcription (germline IH-S-CH transcription), which critically mediates CSR
Primary and secondary stimuli also synergizes to induce AID, whose expression is restricted in B cells undergoing CSR and SHM by positive and negative transcription factors (Figure 3). CD40 engagement and dual TLR-BCR engagement can activate both the canonical and the non-canonical NF-κB pathways for sustained AID gene transcription. In addition to NF-κB, which is ubiquitously expressed and broadly regulates many genes, transcription factors that are expressed in a B cell lineage- and/or differentiation stage-specific fashion regulate AID induction. Prominent among these is HoxC4, an evolutionarily conserved homeodomain transcription factor that is highly expressed in B cells and is induced by the same stimuli that induce AID and CSR. HoxC4 directly binds to the AID gene promoter, through a site (5'-ATTT-3') embedded with a binding site for POU domain-containing transcription factor OCT1/OCT2 (5'-ATTTGAAT-3'), and synergizes with OCT1/OCT2, NF-κB and Sp1/Sp3 to induce AID, thereby inducing CSR and SHM in the antibody response (Park et al., Nature Immunol. 10: 540-550, 2009).
AID is essential for both CSR and SHM; it deaminates deoxycytosines in S and V(D)J region DNA for CSR and SHM, respectively, yielding deoxyuracils. Processing of deoxyuracils by uracil DNA glycosylase (Ung) and then apurinic/apyrimidinic endonucleases (APEs), all of which are critical elements of the base excision repair (BER) pathway, leads to DNA cleavage (Casali and Zan, Nature Immunol. 5: 1101-1103, 2004). In CSR, AID- and Ung-mediated DNA cleavage results in insertion, in the donor and acceptor S regions, of double-strand DNA breaks (DSBs), which are resolved to give rise to S-S junctions and extrachromosomal DNA switch circles
In SHM, it results in resection of blunt DSBs in V(D)J region DNA to generate staggered DSBs, resolving of which by error-prone DNA repair gives rise to mutations (Zan et al., Immunity 2003, 18: 727-738). AID enzymatic activity is inhibited by iron in a highly specific and sensitive manner; it is inhibited by Fe2+, but not other bivalent metal ions, at a narrow range of concentrations (2 - 8 μM) that fall within the putative range of intracellular free iron levels (0.5 - 10 μM), leading to inhibition of CSR in B cells (Li et al., J. Biol. Chem. 287: 21520-21529, 2012).
AID targeting to the Ig loci needs to be tightly regulated to avoid deleterious genome-wide mutations and chromosomal translocations that can lead to neoplastic transformation of both B cells and non-B cells. As we have shown, the core of all S regions is highly enriched in 5'-AGCT-3' repeats. These account for over 45% of Sμ core DNA but less than 1.4% of DNA in the genome (Xu et. al., Nature Struct. Mol. Biol. 17: 1124-1135, 2010). This characteristic is conserved across species that use CSR to diversify their antibodies, from frogs to humans, and would have an evolutionary benefit to CSR
These 5'-AGCT-3' repeats are specifically bound by 14-3-3 adaptors, a family of seven evolutionarily conserved and highly homologous adaptors that in turn target AID to S region DNA through direct protein-protein interactions to unfold CSR (Movie). Targeting of 14-3-3 adaptors and, therefore, AID to only the donor and acceptor S regions, is made possible by the open chromatin state of those S regions as enabled by germline IH-S-CH transcription, and combinatorial histone modifications (Li et. al., Cell Rep. 5: 702-714, 2013), particularly Lys9 acetylation Ser10 phosphorylation of histone 3 (H3K9acS10ph). In addition to 14-3-3 adaptors, which are proper scaffolding proteins, TLS polymerase Rev1 also functions as a scaffolding protein to recruit Ung to S regions
In its role in CSR, Rev1 acts as a structural element, rather than an enzymatic element, uncovering a new role for TLS polymerases in the dynamics of the IgH locus (Zan et al., Cell Rep. 2: 1220-1232, 2012). Overall, targeting of AID, Ung and likely other elements of the SHM and CSR machineries depends on epigenetic modifications of the Ig loci and specific scaffolding proteins
Epigenetic marks, such as DNA methylation, histone posttranslational modifications and microRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating SHM, CSR and differentiation to plasma cells or long-lived memory B cells. Histone modifications together with DNA methylation modulate the expression of critical elements of that machinery, such as AID, as well as factors central to plasma cell differentiation, such as Blimp-1, thereby instructing the maturation of antibody responses (Li et al., Trends in Immunology, 34: 460-470, 2013). Expression of the Aicda (which encodes AID) and Prdm1 (which encodes Blimp-1) genes is also specifically regulated by microRNAs (Zan and Casali, Autoimmunity 46: 81-99, 2013; Li et al., Trends in Immunology, 34: 460-470, 2013). This has led us to investigate the use of histone deacetylase (HDAC) inhibitors (HDIs), which can selectively target certain HDACs, in modulating CSR (Table 1). As indicated by our most recent data, a major regulation mechanism of HDIs is through upregulation of microRNAs that regulate Aicda and Prdm1 transcript levels.
AID expression and SHM and CSR are dysregulated in patients with systemic lupus erythematosus, a debilitating disease that primarily affects women after puberty (Zan et al., Autoimmunity, 42: 89-103, 2009). In these patients, high-affinity and class-switched autoantibodies are generated at high levels and mediate multi-organ damages (Elkon and Casali, Nature Rev. Rheum. 4: 491-498, 2008). As we have shown, dysregulation of AID in lupus B cells (from mouse models of lupus) depends on HoxC4, whose induction by CD40 or TLRs is potentiated by estrogens, which exacerbate disease symptoms of lupus (White et al., Autoimmunity 44: 585-598, 2011). Upon activation, estrogen receptors bind to the three evolutionarily conserved and cooperative estrogen response elements (EREs) we have identified in the HoxC4 gene promoter (Mai et al., J. Biol. Chem. 285: 37797-37810, 2010), thereby upregulating AID in normal B cells for heightened antibody responses and in lupus B cells for the generation of pathogenic autoantibodies
Thus, HoxC4, AID, SHM and CSR are rational targets for therapeutics, particularly small molecules that are already approved by FDA for other diseases.
Recently, we have addressed the B cell-intrinsic role of Rab7, a small GTPase involved in intracellular membrane functions, in antibody responses and underlying mechanisms by constructing conditional knockout Igh+/Cg1-creRab7fl/fl mice. These mice displayed normal B cell and T cell development, as expected, and were deficient in Rab7 only in B cells undergoing IghCg1-cre Ig1-Sg1-Cg1-cre transcription, which, like IgH (Igh+) germline Ig1-Sg1-Cg1 transcription, was induced by IL-4 plus a primary AID- and CSR-inducing stimulus. As these stimuli together induce CSR to IgG1 and IgE, Igh+/Cg1-creRab7fl/fl mice could not mount IgG1 or IgE responses to T-independent and T-dependent antigens, but maintained normal levels of IgM, other IgG isotypes or IgA. Activated Igh+/Cg1-creRab7fl/fl B cells failed to undergo CSR or differentiate into class-switched antibody forming cells, but showed normal proliferation, survival and plasma cell differentiation. This defect was underpinned by the impairment specifically in canonical NF-kB activation and AID induction. To further address the role of Rab7 in autoantibody responses, we have first generated Tg(Aicda-cre)Rab7fl/fl mice on the C57BL/6 background and then bred them onto the MRL/Faslpr/lpr background. We have chosen to go beyond Igh+/Cg1-cre mice to be able to analyze the impact of Rab7 or ERa deficiency on all switched Ig isotypes, not merely IgG1 and IgE. In Tg(Aicda-cre)Rab7fl/fl B cells, Cre expression is under the same strict transcriptional control as Aicda, i.e., in B cells only and only in B cells induced to express AID to undergo CSR/SHM (e.g., upon stimulation by CD154 plus IL-4). Induced C57BL/6Tg(Aicda-cre)Rab7fl/fl B cells will express AID and Cre from the transcriptionally active endogenous Aicda locus and Tg(Aicda-cre), respectively. However, due to Cre expression and consequent deletion of Rab7 (which, as our preliminary data suggest, is itself important for Aicda as well as Aicda-cre induction), these B cells cannot sustain AID expression (Figure 10). This unique tool will allow us to dissect the role of Rab7 in NF-kB activation and Dicer degradation, thereby promoting HoxC4 and AID dysregulation and, ultimately autoantibody responses associated with lupus.