Ig Somatic Hypermutation Research Articles

Antibody Affinity Maturation in Fishes-Our Current Understanding.

It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes.

Atg7 is critical for the functional maturation of germinal center B cells (LYM7P.625)

Abstract The role of autophagy, especially individual components of the autophagy machinery, in antibody response has not been well established. Atg7 is an integral part in autophagesome formation and elongation. Using lineage- and stage-specific conditional knockout (CKO) mice, we have found that, in GC B cell-specific Atg7 CKO mice, although GC formation and structure were not affected by Atg7 deficiency, both primary and memory antibody responses were severely impaired in Atg7 CKO mice. Class-switch recombination (CSR) and Ig somatic hypermutation (SHM) were defective in Atg7-/- GC B-cells, leading to a severely inhibited production of class-switched high-affinity antibodies. Analysis of GC B-cell clones showed that Atg7-deficiency in GC B-cells causes a dramatic change in Ig gene composition in the responding B-cell repertoire, which is dominated by non-canonical Ig genes encoding lower affinity antibodies. Thus, our study demonstrated for the first time that a single gene mutation affects GC function without inhibiting GC formation. The underlying molecular mechanism for impaired GC function in Atg7 mutant mice is that Atg7 is critical in regulating activation-induced deaminase (AID). Atg7-deficiency in GC B cells led to delayed onset and reduced disease severity in collagen-induced arthritis (CIA), demonstrating that Atg7 is critical in the development and pathogenesis of autoimmune disease.

Checkpoint kinase 2 is required for efficient immunoglobulin diversification

Maintenance of genome integrity relies on multiple DNA repair pathways as well as on checkpoint regulation. Activation of the checkpoint kinases Chk1 and Chk2 by DNA damage triggers cell cycle arrest and improved DNA repair, or apoptosis in case of excessive damage. Chk1 and Chk2 have been reported to act in a complementary or redundant fashion, depending on the physiological context. During secondary immunoglobulin (Ig) diversification in B lymphocytes, DNA damage is abundantly introduced by activation-induced cytidine deaminase (AID) and processed to mutations in a locus-specific manner by several error-prone DNA repair pathways. We have previously shown that Chk1 negatively regulates Ig somatic hypermutation by promoting error-free homologous recombination and Ig gene conversion. We now report that Chk2 shows opposite effects to Chk1 in the regulation of these processes. Chk2 inactivation in B cells leads to decreased Ig hypermutation and Ig class switching, and increased Ig gene conversion activity. This is linked to defects in non-homologous end joining and increased Chk1 activation upon interference with Chk2 function. Intriguingly, in the context of physiological introduction of substantial DNA damage into the genome during Ig diversification, the 2 checkpoint kinases thus function in an opposing manner, rather than redundantly or cooperatively.

B Cell–Specific Deficiencies in mTOR Limit Humoral Immune Responses

Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP-specific or anti-Pn14-specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A-specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell-specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.

IL-21 and CD40L Synergistically Promote Plasma Cell Differentiation through Upregulation of Blimp-1 in Human B Cells

After undergoing Ig somatic hypermutation and Ag selection, germinal center (GC) B cells terminally differentiate into either memory or plasma cells (PCs). It is known that the CD40L and IL-21/STAT3 signaling pathways play critical roles in this process, yet it is unclear how the B cell transcription program interprets and integrates these two types of T cell-derived signals. In this study, we characterized the role of STAT3 in the GC-associated PC differentiation using purified human tonsillar GC B cells and a GC B cell-like cell line. When primary GC B cells were cultured under PC differentiation condition, STAT3 inhibition by AG490 prevented the transition from GC centrocytes to preplasmablast, suggesting that STAT3 is required for the initiation of PC development. In a GC B cell-like human B cell line, although IL-21 alone can induce low-level Blimp-1 expression, maximum Blimp-1 upregulation and optimal PC differentiation required both IL-21 and CD40L. CD40L, although having no effect on Blimp-1 as a single agent, greatly augmented the amplitude and duration of IL-21-triggered Jak-STAT3 signaling. In the human PRDM1 locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation.

Critical Analysis of Strand-Biased Somatic Mutation Signatures in TP53versusIg Genes, in Genome-Wide Data and the Etiology of Cancer

Previous analyses of rearranged immunoglobulin (Ig) variable genes (VDJs) concluded that the mechanism of Ig somatic hypermutation (SHM) involves the Ig pre-mRNA acting as a copying template resulting in characteristic strand biased somatic mutation patterns at A:T and G:C base pairs. We have since analysed cancer genome data and found the same mutation strand-biases,in totoor in part, in nonlymphoid cancers. Here we have analysed somatic mutations in a single well-characterised gene TP53. Our goal is to understand the genesis of the strand-biased mutation patterns in TP53—and in genome-wide data—that may arise by “endogenous” mechanisms as opposed to adduct-generated DNA-targeted strand-biased mutations caused by well-characterised “external” carcinogenic influences in cigarette smoke, UV-light, and certain dietary components. The underlying strand-biased mutation signatures in TP53, for many non-lymphoid cancers, bear a striking resemblance to the Ig SHM pattern. A similar pattern can be found in genome-wide somatic mutations in cancer genomes that have also mutated TP53. The analysis implies a role for base-modified RNA template intermediates coupled to reverse transcription in the genesis of many cancers. Thus Ig SHM may be inappropriately activated in many non-lymphoid tissues via hormonal and/or inflammation-related processes leading to cancer.

Minimal Residual Disease Stratification in Fludarabine Ccyclophosphamide Rituximab (FCR) Therapy in Chronic Lymphocytic Leukaemia (CLL

AbstractAbstract 1791Six courses of FCR is considered standard therapy for fit, non-(17p) deleted patients with Chronic Lymphocytic Leukaemia (CLL), however the optimal dose of Rituximab in combined chemotherapy for CLL or the number of FCR courses required has never been formally assessed. Minimal residual disease (MRD) eradication (assessed by 6 colour flow-cytometry(FC) is associated with an increased disease free interval and is a logical endpoint in determining length/efficacy of treatment. Serial MRD testing following therapy provides an objective test of disease re-emergence.A multi-centre prospective phase II study in treatment-naïve CLL patients with modified FCR (Rituximab 375mg/m2) using MRD to determine treatment length/efficacy and identify disease emergence recruited from 2009–2012. Standard pre-treatment assessment plus C-T scan, FISH and Ig somatic hypermutation (SHM) analyses were performed. Patient in radiological and MRD-ve remission after 4 courses of FCR stopped therapy and the remainder received 6 courses. All were followed by 6 monthly MRD assessment.Fifty-two patients were included (35M/17F), mean age 52 years (range 37–72), mean WCC 51 × 109/L (range 8–386), elevated LDH 23 of 45 (51%), lymphadenopathy 43 (83%) and hepatosplenomegaly in 37 (71%). Abnormal FISH results were, del(11q) in 15, +12 in 5, del(13q) in 18; SHM 15(29%) mutated and 37(71%) unmutated or with V3–21 gene usage. Post-treatment MRD is available in 43 patients; 36 (70%) were MRD –ve including 18 (42%) after 4 courses. Nine patients did not complete treatment (toxicity −7, progression-1, non-compliance-1). With a mean follow-up of 20.7 months (range 4.5–38), 6 patients have reverted to MRD +ve at a mean of 13 months (range 10–19) from therapy; only the patient with primary treatment failure has required further chemotherapy. Myelotoxoicty resulted in 23 NCI grade 3 episodes and 7 treatment delays of a mean duration of 25 days (range15–32). One further grade 3 toxicity of pneumonia was identified.Modified FCR was effective in this patient cohort with high risk features (38% unfavourable FISH, 71% unfavourable SHM), with 70% patients achieving MRD-ve status on completion of therapy. 42% of patients became MRD-ve after 4 courses of FCR, suggesting that some patients may not require 6 courses of therapy. Myelotoxicity remains an issue with 7 patients not completing therapy. Longer follow-up will clarify whether shortened therapy will have an impact on MRD+ve reversion, time to re-treatment and survival. Disclosures:No relevant conflicts of interest to declare.

Ultra-Deep Sequencing of De Novo IGHV Mutations in Activated CLL Cells: Evidence for Activation-Induced Deaminase Function.

AbstractAbstract 2545B-cell chronic lymphocytic leukemia (CLL) clones often acquire new mutations and cytogenetic aberrations over time. In other human cancers, including lymphomas and solid tumors, activation-induced deaminase (AID), which normally causes immunoglobulin (Ig) somatic hypermutation (SHM) and isotype class switch recombination (CSR) in germinal center B cells, is expressed and functions abnormally to cause mutations promoting aggressiveness. In CLL, AID mRNA expression in the leukemic cells correlates with increased adverse cytogenetic aberrations and worse clinical outcomes. Furthermore, CLL cells activated by culture with CD32-transfected murine L cells, anti-CD40 and interleukin-4, produce AID protein with associated functions: DNA breaks, Ig CSR, and Ig heavy chain (IGH) variable (IGHV) gene SHM. To evaluate AID-mediated SHM in CLL more accurately, ultra-deep sequencing was performed on CLL clone’s IGH cDNA prior to and after in vitro activation in one unmutated CLL (U-CLL) case (CLL1278, 0.0% mutated IGHV3–30) and one mutated CLL (M-CLL) case (CLL1299, 4.9% mutated IGHV3–23). Additionally, to examine activation of CLL IGH mutation in vivo, ultra-deep sequencing was performed on cells from one U-CLL case (CLL1083, 0.0% mutated IGHV4-b) prior to and after adoptive transfer into the NOD/SCID/γcnull (NSG) mouse, a xenograft model of CLL, where upregulation of AID protein occurs in CD5+CD19+ human CLL cells.Libraries were created for ultra-deep sequencing using the 454 FLX system (Roche) by PCR amplification with IGHV family-specific framework1 (Lprimer) and IGH constant region Cμ (IGHM) (Rprimer) primers on cDNA obtained from CLL cells prior to (day 0) or after in vitro culture for 7 (CLL1278) or 14 days (CLL1278; CLL1299) or from NSG spleen CLL cells collected 35 days after transfer (CLL1083). The resulting 461,153 sequence reads were processed to generate separate datasets with fixed sequence block lengths for each primer. The Lprimer sequence blocks included only 5’IGHV sequence, while the Rprimer sequence blocks encompassed 3’IGHV, IGH diversity, and IGH joining genes (IGHVDJ) as well as 5’IGHM sequence. Individual subclone sequences that occurred at least twice were extracted from each of the datasets and the unique de novo subclones not shared between day 0 and activation were analyzed for mutations.All three CLL cases showed increases in 5'IGHV and 3'IGHVDJ subclones with activation. After in vitro activation, for CLL1278, 123,518 total sequence reads produced 68 unique subclones as compared to 33 at day 0; and for CLL1299, 163,358 total sequence reads produced 78 unique subclones as compared to 61 at day 0. Likewise, after in vivo activation in the NSG mouse, for CLL1083, 174,472 total sequence reads produced 91 unique subclones as compared to 56 at day 0. In contrast, all three CLL cases showed decreases in 5'IGHM subclones after activation. After in vitro activation, CLL1278 and CLL1299 decreased from 22 and 20 unique day 0 subclones to 13 and 16 unique subclones. Similarly, CLL1083 showed a decrease from 20 unique day 0 subclones to 11 unique subclones after transfer into the NSG mouse. After normalization for read number and block sequence length, all three CLL cases showed an increase in 5'IGHV mutation with CLL cell activation (fold change relative to 5'IGHM = 3.4, 2.2, and 4.6 for CLL1278, CLL1299, and CLL1083, respectively). This increase in IGHV mutation relative to IGHM following activation is consistent with AID activity. Furthermore, examination of mutation sites in these subclones revealed an increase in mutations in AID hotspot motifs (GYW or WRC) in the 5'IGHV of all three CLL cases with activation (fold change = 2.0, 1.9, and 2.5 for CLL1278, CLL1299, and CLL1083, respectively), which was not observed further downstream in 3'IGHVDJ and 5'IGHM.Thus, by analyzing a very large number of IGH sequences in CLL cells after activation in vitro or in vivo, a pattern of de novo mutations consistent with AID activity is found. Furthermore, since both U-CLL and M-CLL clones exhibited AID activity, these findings indicate that AID-mediated SHM is not limited by CLL IGHV mutation status. Finally, these data support a model of AID-promoted mistargeted mutations, which may lead to adverse cytogenetic aberrations and unfavorable outcomes in CLL. Disclosures:No relevant conflicts of interest to declare.

2.6 Anti-Inflammatory Curcumin Inhibits Activation-Induced Cytosine Deaminase (AID) within Cycling Normal and CLL B Lymphocytes

Abstract Abstract 4606 Curcumin is a natural phenolic compound within the spice, Curcuma longa. It has noted anti-inflammatory effects, in large part due to its potent suppressive effect on the NF-kB signaling pathway. AID is a NF-kB-regulated enzyme, essential for B cell Ig class switch recombination and somatic hypermutation and recently shown to promote oncogenic transformation within both B cell and non-B cell lineages. This study has examined the effect of curcumin on the division-linked upregulation of AID protein and mRNA within several human B cell populations: in vitro-activated normal and CLL B lymphocytes and the AID-positive, pre-germinal center B cell line, CL-01. CFSE-labeled, IgM+ human B2 cells isolated from spleen/tonsil were pre-activated for 4–5 days with stimuli likely encountered in sites of inflammation, i.e. limiting surrogate C3dg-coated antigen (anti-IgM: anti-CD21: dextran) + IL-4 + BAFF. Peripheral blood B-CLL cells were activated with TLR-9 ligand, ODN-2006, + IL-15. Curcumin at doses from 6 to 50 μ M, and parallel DMSO vehicle controls, were pulsed into dividing B cell cultures (day 3, 4, or 5 of activation), and AID mRNA and protein assessed after 1 to 2 days. In experiments with CL-01 B cells, the kinetics of curcumin-induced AID suppression was further analyzed. Messenger RNA was monitored by both quantitative and qualitative RT-PCR; AID protein was assessed by two-color flow cytometry of CFSE-labeled cells and immunoblotting. The above experiments revealed that curcumin can significantly down-regulate AID mRNA and protein, in dose dependent fashion within each of the above B cell populations. Following a 16h pulse of curcumin (25 μ M), AID mRNA within CL-01 cells was inhibited by 60% (p=0.001), and accompanied by ~ 60% decrease in AID protein. Within cultures of replicating normal human B lymphocytes, a similar pulse of curcumin reduced total culture AID mRNA by an average of 70% in 3 experiments. AID protein in blasts representing 3–4 divisions was reduced by 79%, and in those representing 1–2 divisions by 58%, within a representative experiment. AID mRNA was evident within all in vitro-activated B-CLL clones tested (total = 6 clones at the time of submission). This was significantly reduced by a 15–24 hr pulse with curcumin (20-25 μ M): 42% inhibition (p=0.02)). The inhibitory effects of curcumin were evident in both IgHV mutated and unmutated clones. Within stimulated B-CLL assessed for AID protein (4 total clones, of which 2 were positive), a 20 hr pulse of curcumin at 40 μ M and 20 μ M reduced AID expression in one clone by 80% and 40%, respectively. In the other clone, a maximal tested dose of 20 μ M curcumin reduced AID protein by only 12%. Suppression of AID mRNA in the CL-01 cell line was noted as soon as 3 hours following exposure to curcumin and was preceded by inhibition of NF-kB activation, both baseline and BAFF-induced. The latter was determined through monitoring intracellular levels of phospho-p65-Ser(529) – an active phosphorylated form of NF-kB RelA. Taken together, these findings suggest that NF-kB- and AID-suppressing curcumin may be useful in reducing the risk of malignant transformation and B-CLL progression into more malignant subclones, as well as treating B cell autoimmune diseases driven by pathogenic, somatically-mutated IgG autoantibodies. Disclosures: No relevant conflicts of interest to declare.

Translocation-Capture Sequencing Reveals the Extent and Nature of Chromosomal Rearrangements in B Lymphocytes

Chromosomal rearrangements, including translocations, require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are frequently involved in producing leukemias, lymphomas and sarcomas. Despite the importance of these events, current understanding of their genesis is limited. To examine the origins of chromosomal rearrangements we developed Translocation Capture Sequencing (TC-Seq), a method to document chromosomal rearrangements genome-wide, in primary cells. We examined over 180,000 rearrangements obtained from 400 million B lymphocytes, revealing that proximity between DSBs, transcriptional activity and chromosome territories are key determinants of genome rearrangement. Specifically, rearrangements tend to occur in cis and to transcribed genes. Finally, we find that activation-induced cytidine deaminase (AID) induces the rearrangement of many genes found as translocation partners in mature B cell lymphoma.

The Eleven-Nineteen Lysine-rich Leukemia Gene (ELL2) Influences the Histone H3 Protein Modifications Accompanying the Shift to Secretory Immunoglobulin Heavy Chain mRNA Production

In plasma cells, immunoglobulin heavy chain (IgH) secretory-specific mRNA is made in high abundance as a result of both increased promoter proximal poly(A) site choice and weak splice-site skipping. Ell2, the eleven-nineteen lysine rich leukemia gene, is a transcription elongation factor that is induced ∼6-fold in plasma cells and has been shown to drive secretory-specific mRNA production. Reducing ELL2 by siRNA, which reduced processing to the secretion-specific poly(A) site, also influenced the methylations of histone H3K4 and H3K79 on the IgH gene and impacted positive transcription factor b (pTEFb), Ser-2 carboxyl-terminal phosphorylation, and polyadenylation factor additions to RNA polymerase II. The multiple lineage leukemia gene (MLL) and Dot1L associations with the IgH gene were also impaired in the absence of ELL2. To investigate the link between histone modifications, transcription elongation, and alternative RNA processing in IgH mRNA production, we performed chromatin immunoprecipitation on cultured mouse B and plasma cells bearing the identical IgH γ2a gene. In the plasma cells, as compared with the B cells, the H3K4 and H3K79 methylations extended farther downstream, past the IgH enhancer to the end of the transcribed region. Thus the downstream H3K4 and H3K79 methylation of the IgH associated chromatin in plasma cells is associated with increased polyadenylation and exon skipping, resulting from the actions of ELL2 transcription elongation factor.

Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways

AbstractMultiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27−IgG+ and CD27+IgM+ B cells are derived from primary germinal center reactions, and CD27+IgA+ and CD27+IgG+ B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27−IgA+ memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27−IgA+ cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases.

Foxo1 is essential for germinal center B cell functions and the development of collagen-induced arthritis (101.15)

Abstract Foxo1 has been shown to be critical for B-cell lymphopoiesis by regulating several genes involved in immunoglobulin (Ig) rearrangement. However, the role of Foxo1 in germinal center (GC) B cell functions remains unclear. Unlike being downregulated in activated B cells, Foxo1 expression is elevated in GC B cells, suggesting a distinctive regulatory role. In Foxo1 GC B-cell conditional knockout (CKO) mice, both primary and memory antibody responses are severely impaired after immunization with T-dependent antigen NP-CGG. The numbers of NP-specific IgG1-secreting B-cells, as well as the NP-specific IgG1 antibody titer in the serum, are dramatically reduced in the mutant mice. The results indicate an inability to undergo Ig class-switch recombination (CSR) in Foxo1-deficient GC B cells, possibly due to the abolished expression of activation-induced cytidine deaminase. In addition, analysis of mutation rate of Ig genes in the NP-specific B cells reveals that Ig somatic hypermutation (SHM) is impaired in the Foxo1-deficient GC B cells. The clonotypes of B cells responding to NP are also altered in the mutant mice. Remarkably, using collagen-induced arthritis model, we have found that Foxo1 GC B-cell CKO mice are resistant to arthritis induction. Taken together, our data indicate that Foxo1 is essential for normal antibody responses, CSR and SHM of GC B cells. The absence of Foxo1 in GC B cells prevents the development of autoimmune arthritis.

The dependence of Ig class‐switching on the nuclear export sequence of AID likely reflects interaction with factors additional to Crm1 exportin

Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that – beyond a requirement for threshold Crm1 binding – there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1.

AID expression during B-cell development: searching for answers

Expression of activation-induced cytidine deaminase (AID) by germinal center (GC) B cells drives the processes of immunoglobulin (Ig) somatic hypermutation (SHM) and class switch recombination (CSR) necessary for the generation of high affinity IgG serum antibody and the memory B-cell compartment. Increasing evidence indicates that AID is also expressed at low levels in developing B cells but to date, this early, developmentally regulated AID expression has no known function. Does the timing and extent of AID expression in developmentally immature, non-GC B cells provide clues to reveal its physiologic role?

Anti-Inflammatory Curcumin Inhibits Activation-Induced Cytosine Deaminase (AID) within Cycling Normal and CLL B Lymphocytes.

AbstractAbstract 4606Curcumin is a natural phenolic compound within the spice, Curcuma longa. It has noted anti-inflammatory effects, in large part due to its potent suppressive effect on the NF-kB signaling pathway. AID is a NF-kB-regulated enzyme, essential for B cell Ig class switch recombination and somatic hypermutation and recently shown to promote oncogenic transformation within both B cell and non-B cell lineages. This study has examined the effect of curcumin on the division-linked upregulation of AID protein and mRNA within several human B cell populations: in vitro-activated normal and CLL B lymphocytes and the AID-positive, pre-germinal center B cell line, CL-01. CFSE-labeled, IgM+ human B2 cells isolated from spleen/tonsil were pre-activated for 4–5 days with stimuli likely encountered in sites of inflammation, i.e. limiting surrogate C3dg-coated antigen (anti-IgM: anti-CD21: dextran) + IL-4 + BAFF. Peripheral blood B-CLL cells were activated with TLR-9 ligand, ODN-2006, + IL-15. Curcumin at doses from 6 to 50 μ M, and parallel DMSO vehicle controls, were pulsed into dividing B cell cultures (day 3, 4, or 5 of activation), and AID mRNA and protein assessed after 1 to 2 days. In experiments with CL-01 B cells, the kinetics of curcumin-induced AID suppression was further analyzed. Messenger RNA was monitored by both quantitative and qualitative RT-PCR; AID protein was assessed by two-color flow cytometry of CFSE-labeled cells and immunoblotting. The above experiments revealed that curcumin can significantly down-regulate AID mRNA and protein, in dose dependent fashion within each of the above B cell populations. Following a 16h pulse of curcumin (25 μ M), AID mRNA within CL-01 cells was inhibited by 60% (p=0.001), and accompanied by ~ 60% decrease in AID protein. Within cultures of replicating normal human B lymphocytes, a similar pulse of curcumin reduced total culture AID mRNA by an average of 70% in 3 experiments. AID protein in blasts representing 3–4 divisions was reduced by 79%, and in those representing 1–2 divisions by 58%, within a representative experiment. AID mRNA was evident within all in vitro-activated B-CLL clones tested (total = 6 clones at the time of submission). This was significantly reduced by a 15–24 hr pulse with curcumin (20-25 μ M): 42% inhibition (p=0.02)). The inhibitory effects of curcumin were evident in both IgHV mutated and unmutated clones. Within stimulated B-CLL assessed for AID protein (4 total clones, of which 2 were positive), a 20 hr pulse of curcumin at 40 μ M and 20 μ M reduced AID expression in one clone by 80% and 40%, respectively. In the other clone, a maximal tested dose of 20 μ M curcumin reduced AID protein by only 12%. Suppression of AID mRNA in the CL-01 cell line was noted as soon as 3 hours following exposure to curcumin and was preceded by inhibition of NF-kB activation, both baseline and BAFF-induced. The latter was determined through monitoring intracellular levels of phospho-p65-Ser(529) – an active phosphorylated form of NF-kB RelA. Taken together, these findings suggest that NF-kB- and AID-suppressing curcumin may be useful in reducing the risk of malignant transformation and B-CLL progression into more malignant subclones, as well as treating B cell autoimmune diseases driven by pathogenic, somatically-mutated IgG autoantibodies. Disclosures:No relevant conflicts of interest to declare.

Anti-inflammatory curcumin inhibits AID expression within cycling human B cells (96.21)

Abstract Curcumin is a natural phenolic compound extracted from the spice, Curcuma longa. It has noted anti-inflammatory effects, in part due to its suppression of NF-kB. Activation-induced cytosine deaminase (AID) is a NF-kB-regulated enzyme essential for Ig class switch recombination and somatic hyper-mutation with a demonstrated role in lymphomagenesis. This study has examined the effect of curcumin on the division-dependent upregulation of AID protein and mRNA within in vitro-activated normal human B lymphocytes and the AID-expressing B cell line, CL-01. CFSE-labeled, IgM+ human B2 cells isolated from spleen/tonsil were pre-activated for 4-5 days with stimuli likely encountered in sites of inflammation, i.e. limiting surrogate C3dg-coated antigen (anti-IgM: anti-CD21: dextran) + IL-4 + BAFF. Curcumin at doses from 6 to 50 µM, or parallel vehicle control, was pulsed into dividing B cell cultures and AID mRNA and protein were assessed after 1 or 2 d, respectively. Expression of mRNA was monitored by both quantitative and qualitative RT-PCR. AID protein was assessed, by two-color flow cytometry of CFSE-labeled cells and immunoblotting. Curcumin significantly down-regulated AID mRNA and protein, in dose dependent fashion. Accompanying this decline was a diminished recovery of IgG+ class-switched B cells within the divided population. This study suggests that curcumin could have a role in treating B cell autoimmune disease and reducing the risk of malignant transformation.

Evidence for Antigen-Driven Development of Molecularly Classified Burkitt Lymphomas.

Abstract 317 Evidence for Antigen-driven Development of Molecularly Classified Burkitt LymphomasThe presence of somatic mutations and intraclonal diversity (ID) in the variable region of rearranged immunoglobulin (Ig) genes is commonly used as a marker of the cellular passage of B cells through the germinal center (GC) reaction in secondary lymphoid tissues following an in vivo antigen encounter. Such features have also been documented in different subgroups of aggressive B-cell lymphomas, suggesting that antigen selection pressure has taken place during lymphoma development. We undertook a detailed analysis of the Ig repertoire and somatic hypermutation (SHM) status of 54 molecularly classified Burkitt lymphomas (BL) (Hummel et al. 2006). The IGHV gene repertoire was remarkably biased, with only three genes accounting for 40.8% of all cases (IGHV4-34, 18.5%; IGHV4-39, 11.1%; IGHV3-30, 11.1%). Of note, this bias was even more evident when cases were grouped according to age < or > 14 years, with the IGVH4-34 and IGVH4-39 rearrangements occurring almost exclusively in pediatric BL (7/11 and 5/6 cases respectively). Only 2/54 cases (3.7%) carried unmutated IGHV genes (100% germline identity, GI); 12/54 cases (22.2%) carried IGHV genes of borderline/minimally mutated status (98–99.9% GI); finally, the remaining 40/54 cases (74.1%) carried mutated IGHV genes (<98% GI). The median GI of the cohort was 95.2% (range: 86.4–100%) with a significantly different mutation load according to age below or above 14 years (GI 95.8 vs. 92.9 respectively, p=0.0168). Shared replacement mutations (“stereotyped” amino acid (AA) changes) at certain codon positions were identified in 12/54 (22.2%) BL cases. Stereotyped AA changes were found only amongst rearrangements utilizing the IGHV4-34 and IGHV4-39 genes, strongly indicating evidence for an antigen driven SHM process in BL. The majority of stereotyped AA changes identified here were distinct from those previously reported in other entities, in particular CLL, and thus can be considered as BL-biased. Also, of note, the IGVH4-34 specific motif responsible for binding the N-acetyl-lactosamine antigenic determinant was found intact in 11 IGVH4-34 BL sequences, implying that these clones retained the ability to bind to and be activated by superantigens. Furthermore, the distribution of mutations was different in IGHV4-34 vs. non-IGHV4-34 cases, as evidenced by low R-to-S ratios in the heavy complementarity-determining regions (especially HCDR1), indicating a different selection pattern of VH-genes according to varying antigens.To assess the presence of ID through ongoing SHM, we evaluated 411 subcloned sequences from 11 IGVH4-34 and 6 IGVH4-39 cases. Only one case carried identical sets of subcloned sequences; seven cases carried mutations in single subcloned sequences that were considered as unconfirmed; finally, 9 cases carried at least one confirmed mutation among two or more subcloned sequences and, thus, were considered as exhibiting confirmed ID. However, analysis of the distribution of the so called unconfirmed changes provided further evidence for the very precise targeting of mutations introduced as part of the ID process. Thus, certain changes identified in single subcloned sequences from one case were shared by most or all subcloned sequences of another case utilizing the same IGHV gene (“confirmed by another case”). In conclusion, our data demonstrate that BL is characterized by a highly distinctive IG gene repertoire with a precisely targeted SHM process. In addition, they provide strong evidence for a functionally driven SHM, supporting a role for antigen-driven selection of the clonogenic progenitors. Disclosures:No relevant conflicts of interest to declare.

Entertaining but educational immunology book for children

Entertaining but educational immunology book for children

Integrity of the AID serine-38 phosphorylation site is critical for class switch recombination and somatic hypermutation in mice

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AID(S38A)), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AID(WT)). B cells homozygous for the AID(S38A) mutation show substantially impaired CSR and SHM, correlating with inability of AID(S38A) to interact with endogenous RPA. Moreover, mice haploinsufficient for AID(S38A) have even more severely impaired CSR when compared with mice haploinsufficient for AID(WT), with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.