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Antibody diversification is essential for an effective immune response, with somatic hypermutation (SHM) serving as a key molecular process in this adaptation. Activation-induced cytidine deaminase (AID) initiates SHM by inducing DNA lesions, which are ultimately resolved into point mutations, as well as small insertions and deletions (indels). These mutational outcomes contribute to antibody affinity maturation. The mechanisms responsible for generating point mutations and indels involve the base excision repair (BER) and mismatch repair (MMR) pathways, which are well coordinated to maintain genomic integrity while allowing for beneficial mutations to occur. In this regard, translesion synthesis (TLS) polymerases contribute to the diversity of mutational outcomes in antibody genes by enabling the bypass of DNA lesions. This review summarizes our current understanding of the distinct molecular mechanisms that generate point mutations and indels during SHM. Understanding these mechanisms is critical for elucidating the development of broadly neutralizing antibodies (bnAbs) and autoantibodies, and has implications for vaccine design and therapeutics. [ABSTRACT FROM AUTHOR]

Introduction: Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, longterm humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context. Methods: Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions. Results: DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5'C and 3'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for Ebox transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation. Discussion: Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions. [ABSTRACT FROM AUTHOR]

IntroductionSomatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5’ ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context.MethodsHere, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions.ResultsDeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5’C and 3’G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3’G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation.DiscussionOur results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.

Introduction: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, accounting for 30-40% of all adult leukemias. The dynamics of Blymphocyte CLL clones with mutated immunoglobulin heavy chain variable region (IgHV) genes in their tumor (M-CLL) can be studied using mutational lineage trees. Methods: Here, we used lineage tree-based analyses of somatic hypermutation (SHM) and selection in M-CLL clones, comparing the dominant (presumably malignant) clones of 15 CLL patients to their non-dominant (presumably normal) B cell clones, and to those of healthy control repertoires. This type of analysis, which was never previously published in CLL, yielded the following novel insights. Results: CLL dominant clones undergo - or retain - more replacement mutations that alter amino acid properties such as charge or hydropathy. Although, as expected, CLL dominant clones undergo weaker selection for replacement mutations in the complementarity determining regions (CDRs) and against replacement mutations in the framework regions (FWRs) than non-dominant clones in the same patients or normal B cell clones in healthy controls, they surprisingly retain some of the latter selection in their FWRs. Finally, using machine learning, we show that even the non-dominant clones in CLL patients differ from healthy control clones in various features, most notably their expression of higher fractions of transition mutations. Discussion: Overall, CLL seems to be characterized by significant loosening - but not a complete loss - of the selection forces operating on B cell clones, and possibly also by changes in SHM mechanisms. [ABSTRACT FROM AUTHOR]

Intoduction: Two scaffold/matrix attachment regions (5'- and 3'-MARsEμ) flank the intronic core enhancer (cEμ) within the immunoglobulin heavy chain locus (IgH). Besides their conservation in mice and humans, the physiological role of MARsEμ is still unclear and their involvement in somatic hypermutation (SHM) has never been deeply evaluated. Methods: Our study analyzed SHM and its transcriptional control in a mouse model devoid of MARsEμ, further combined to relevant models deficient for base excision repair and mismatch repair. Results: We observed an inverted substitution pattern in of MARsEμ-deficient animals: SHM being decreased upstream from cEμ and increased downstream of it. Strikingly, the SHM defect induced by MARsEμ-deletion was accompanied by an increase of sense transcription of the IgH V region, excluding a direct transcription-coupled effect. Interestingly, by breeding to DNA repair-deficient backgrounds, we showed that the SHM defect, observed upstream from cEμ in this model, was not due to a decrease in AID deamination but rather the consequence of a defect in base excision repair-associated unfaithful repair process. Discussion: Our study pointed out an unexpected "fence" function of MARsEμ regions in limiting the error-prone repair machinery to the variable region of Ig gene loci. [ABSTRACT FROM AUTHOR]

The analysis of the immunogenetic background of multiple myeloma (MM) has proven key to understanding disease ontogeny. However, limited information is available regarding the immunoglobulin (IG) gene repertoire in MM cases carrying different heavy chain isotypes. Here, we studied the IG gene repertoire in a series of 523 MM patients, of whom 165 and 358 belonged to the IgA and IgG MM groups, respectively. IGHV3 subgroup genes predominated in both groups. However, at the individual gene level, significant (p<0.05) differences were identified regarding IGHV3-21 (frequent in IgG MM) and IGHV5-51 (frequent in IgA MM). Moreover, biased pairings were identified between certain IGHV genes and IGHD genes in IgA versus IgG MM. Turning to the imprints of somatic hypermutation (SHM), the bulk of rearrangements (IgA: 90.9%, IgG: 87.4%) were heavily mutated [exhibiting an IGHV germline identity (GI) <95%]. SHM topology analysis disclosed distinct patterns in IgA MM versus IgG MM cases expressing B cell receptor IG encoded by the same IGHV gene: the most pronounced examples concerned the IGHV3-23, IGHV3-30 and IGHV3-9 genes. Furthermore, differential SHM targeting was also identified between IgAMMversus IgG MM, particularly in cases utilizing certain IGHV genes, alluding to functional selection. Altogether, our detailed immunogenetic evaluation in the largest to-date series of IgA and IgG MM patients reveals certain distinct features in the IGH gene repertoires and SHM. These findings suggest distinct immune trajectories for IgA versus IgG MM, further underlining the role of external drive in the natural history of MM. [ABSTRACT FROM AUTHOR]

IntoductionTwo scaffold/matrix attachment regions (5’- and 3’-MARsEµ) flank the intronic core enhancer (cEµ) within the immunoglobulin heavy chain locus (IgH). Besides their conservation in mice and humans, the physiological role of MARsEµ is still unclear and their involvement in somatic hypermutation (SHM) has never been deeply evaluated.MethodsOur study analyzed SHM and its transcriptional control in a mouse model devoid of MARsEµ, further combined to relevant models deficient for base excision repair and mismatch repair.ResultsWe observed an inverted substitution pattern in of MARsEµ-deficient animals: SHM being decreased upstream from cEµ and increased downstream of it. Strikingly, the SHM defect induced by MARsEµ-deletion was accompanied by an increase of sense transcription of the IgH V region, excluding a direct transcription-coupled effect. Interestingly, by breeding to DNA repair-deficient backgrounds, we showed that the SHM defect, observed upstream from cEµ in this model, was not due to a decrease in AID deamination but rather the consequence of a defect in base excision repair-associated unfaithful repair process.DiscussionOur study pointed out an unexpected “fence” function of MARsEµ regions in limiting the error-prone repair machinery to the variable region of Ig gene loci.

Somatic hypermutation (SHM) is an important diversification mechanism that plays a part in the creation of immune memory. Immunoglobulin (Ig) variable region gene lineage trees were used over the last four decades to model SHM and the selection mechanisms operating on B cell clones. We hereby present IgTreeZ (Immunoglobulin Tree analyZer), a python-based tool that analyses many aspects of Ig gene lineage trees and their repertoires. Using simulations, we show that IgTreeZ can be reliably used for mutation and selection analyses. We used IgTreeZ on empirical data, found evidence for different mutation patterns in different B cell subpopulations, and gained insights into antigendriven selection in corona virus disease 19 (COVID-19) patients. Most importantly, we show that including the CDR3 regions in selection analyses – which is only possible if these analyses are lineage tree-based – is crucial for obtaining correct results. Overall, we present a comprehensive lineage tree analysis tool that can reveal new biological insights into B cell repertoire dynamics. [ABSTRACT FROM AUTHOR]

Somatic hypermutation (SHM) is an important diversification mechanism that plays a part in the creation of immune memory. Immunoglobulin (Ig) variable region gene lineage trees were used over the last four decades to model SHM and the selection mechanisms operating on B cell clones. We hereby present IgTreeZ (Immunoglobulin Tree analyZer), a python-based tool that analyses many aspects of Ig gene lineage trees and their repertoires. Using simulations, we show that IgTreeZ can be reliably used for mutation and selection analyses. We used IgTreeZ on empirical data, found evidence for different mutation patterns in different B cell subpopulations, and gained insights into antigen-driven selection in corona virus disease 19 (COVID-19) patients. Most importantly, we show that including the CDR3 regions in selection analyses – which is only possible if these analyses are lineage tree-based – is crucial for obtaining correct results. Overall, we present a comprehensive lineage tree analysis tool that can reveal new biological insights into B cell repertoire dynamics.

Activated B-cells diversify their antibody repertoire via somatic hypermutation (SHM) and class switch recombination (CSR). SHM is restricted to the variable region, whereas, CSR is confined to the constant region of immunoglobulin (Ig) genes. Activation-induced cytidine deaminase (AID) is a crucial player in the diversification of antibodies in the activated B-cell. AID catalyzes the deamination of cytidine (C) into uracil (U) at Ig genes. Subsequently, low fidelity repair of U:G mismatches may lead to mutations. Transcription is essential for the AID action, as it provides a transient single-strand DNA substrate. Since splicing is a co-transcriptional event, various splicing factors or regulators influence the transcription. Numerous splicing factors are known to regulate the AID targeting, function, Ig transcription, and AID splicing, which eventually influence antibody diversification processes. Splicing regulator SRSF1-3, a splicing isoform of serine arginine-rich splicing factor (SRSF1), and CTNNBL1, a spliceosome interacting factor, interact with AID and play a critical role in SHM. Likewise, a splicing regulator polypyrimidine tract binding protein-2 (PTBP2) and the debranching enzyme (DBR1) debranches primary switch transcripts which later forms G-quadruplex structures, and the S region guide RNAs direct AID to S region DNA. Moreover, AID shows several alternate splicing isoforms, like AID devoid of exon-4 (AIDΔE4) that is expressed in various pathological conditions. Interestingly, RBM5, a splicing regulator, is responsible for the skipping of AID exon 4. In this review, we discuss the role and significance of splicing factors in the AID mediated antibody diversification. [ABSTRACT FROM AUTHOR]

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.

A T‐cell receptor (TCR) with optimal avidity to a tumor antigen can be used to redirect T cells to eradicate cancer cells via adoptive cell transfer. Cancer testis antigens (CTAs) are attractive targets because they are expressed in the testis, which is immune‐privileged, and in the tumor. However, CTAs are self‐antigens and natural TCRs to CTAs have low affinity/avidity due to central tolerance. We previously described a method of directed evolution of TCR avidity using somatic hypermutation. In this study, we made several improvements to this method and enhanced the avidity of the hT27 TCR, which is specific for the cancer testis antigen HLA‐A2‐MAGE‐A1278‐286. We identified eight point mutations with varying degrees of improved avidity. Human T cells transduced with TCRs containing these mutations displayed enhanced tetramer binding, IFN‐γ and IL2 production, and cytotoxicity. Most of the mutations have retained specificity, except for one mutant with extremely high avidity. We demonstrate that somatic hypermutation is capable of optimizing avidity of clinically relevant TCRs for immunotherapy. [ABSTRACT FROM AUTHOR]

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region. [ABSTRACT FROM AUTHOR]

The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring at an estimated rate of 10,000–30,000 per cell/per day, and mostly involves chemical DNA base lesions caused by oxidation, depurination, alkylation, and deamination. The base excision repair (BER) pathway is primary responsible for removing and repairing these small base lesions that would otherwise lead to mutations or DNA breaks during replication. Next to preventing DNA mutations and damage, the BER pathway is also involved in mutagenic processes in B cells during immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM), which are instigated by uracil (U) lesions derived from activation-induced cytidine deaminase (AID) activity. BER is required for the processing of AID-induced lesions into DNA double strand breaks (DSB) that are required for CSR, and is of pivotal importance for determining the mutagenic outcome of uracil lesions during SHM. Although uracils are generally efficiently repaired by error-free BER, this process is surprisingly error-prone at the Ig loci in proliferating B cells. Breakdown of this high-fidelity process outside of the Ig loci has been linked to mutations observed in B-cell tumors and DNA breaks and chromosomal translocations in activated B cells. Next to its role in preventing cancer, BER has also been implicated in immune tolerance. Several defects in BER components have been associated with autoimmune diseases, and animal models have shown that BER defects can cause autoimmunity in a B-cell intrinsic and extrinsic fashion. In this review we discuss the contribution of BER to genomic integrity in the context of immune receptor diversification, cancer and autoimmune diseases.

Somatic hypermutation (SHM) of the immunoglobulin variable (IgV) loci is a key process in antibody affinity maturation. The enzyme activation-induced deaminase (AID), initiates SHM by creating C → U mismatches on single-stranded DNA (ssDNA). AID has preferential hotspot motif targets in the context of WRC/GYW (W = A/T, R = A/G, Y = C/T) and particularly at WGCW overlapping hotspots where hotspots appear opposite each other on both strands. Subsequent recruitment of the low-fidelity DNA repair enzyme, Polymerase eta (Polη), during mismatch repair, creates additional mutations at WA/TW sites. Although there are more than 50 functional immunoglobulin heavy chain variable (IGHV) segments in humans, the fundamental differences between these genes and their ability to respond to all possible foreign antigens is still poorly understood. To better understand this, we generated profiles of WGCW hotspots in each of the human IGHV genes and found the expected high frequency in complementarity determining regions (CDRs) that encode the antigen binding sites but also an unexpectedly high frequency of WGCW in certain framework (FW) sub-regions. Principal Components Analysis (PCA) of these overlapping AID hotspot profiles revealed that one major difference between IGHV families is the presence or absence of WGCW in a sub-region of FW3 sometimes referred to as “CDR4.” Further differences between members of each family (e.g., IGHV1) are primarily determined by their WGCW densities in CDR1. We previously suggested that the co-localization of AID overlapping and Polη hotspots was associated with high mutability of certain IGHV sub-regions, such as the CDRs. To evaluate the importance of this feature, we extended the WGCW profiles, combining them with local densities of Polη (WA) hotspots, thus describing the co-localization of both types of hotspots across all IGHV genes. We also verified that co-localization is associated with higher mutability. PCA of the co-localization profiles showed CDR1 and CDR2 as being the main contributors to variance among IGHV genes, consistent with the importance of these sub-regions in antigen binding. Our results suggest that AID overlapping (WGCW) hotspots alone or in conjunction with Polη (WA/TW) hotspots are key features of evolutionary variation between IGHV genes.

The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring at an estimated rate of 10,000–30,000 per cell/per day, and mostly involves chemical DNA base lesions caused by oxidation, depurination, alkylation, and deamination. The base excision repair (BER) pathway is primary responsible for removing and repairing these small base lesions that would otherwise lead to mutations or DNA breaks during replication. Next to preventing DNA mutations and damage, the BER pathway is also involved in mutagenic processes in B cells during immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM), which are instigated by uracil (U) lesions derived from activation-induced cytidine deaminase (AID) activity. BER is required for the processing of AID-induced lesions into DNA double strand breaks (DSB) that are required for CSR, and is of pivotal importance for determining the mutagenic outcome of uracil lesions during SHM. Although uracils are generally efficiently repaired by error-free BER, this process is surprisingly error-prone at the Ig loci in proliferating B cells. Breakdown of this high-fidelity process outside of the Ig loci has been linked to mutations observed in B-cell tumors and DNA breaks and chromosomal translocations in activated B cells. Next to its role in preventing cancer, BER has also been implicated in immune tolerance. Several defects in BER components have been associated with autoimmune diseases, and animal models have shown that BER defects can cause autoimmunity in a B-cell intrinsic and extrinsic fashion. In this review we discuss the contribution of BER to genomic integrity in the context of immune receptor diversification, cancer and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Somatic hypermutation (SHM) of the immunoglobulin variable (IgV) loci is a key process in antibody affinity maturation. The enzyme activation-induced deaminase (AID), initiates SHM by creating C → U mismatches on single-stranded DNA (ssDNA). AID has preferential hotspot motif targets in the context of WR C / G YW (W = A/T, R = A/G, Y = C/T) and particularly at W GC W overlapping hotspots where hotspots appear opposite each other on both strands. Subsequent recruitment of the low-fidelity DNA repair enzyme, Polymerase eta (Polη), during mismatch repair, creates additional mutations at W A / T W sites. Although there are more than 50 functional immunoglobulin heavy chain variable (IGHV) segments in humans, the fundamental differences between these genes and their ability to respond to all possible foreign antigens is still poorly understood. To better understand this, we generated profiles of WGCW hotspots in each of the human IGHV genes and found the expected high frequency in complementarity determining regions (CDRs) that encode the antigen binding sites but also an unexpectedly high frequency of WGCW in certain framework (FW) sub-regions. Principal Components Analysis (PCA) of these overlapping AID hotspot profiles revealed that one major difference between IGHV families is the presence or absence of WGCW in a sub-region of FW3 sometimes referred to as "CDR4." Further differences between members of each family (e.g., IGHV1) are primarily determined by their WGCW densities in CDR1. We previously suggested that the co-localization of AID overlapping and Polη hotspots was associated with high mutability of certain IGHV sub-regions, such as the CDRs. To evaluate the importance of this feature, we extended the WGCW profiles, combining them with local densities of Polη (WA) hotspots, thus describing the co-localization of both types of hotspots across all IGHV genes. We also verified that co-localization is associated with higher mutability. PCA of the co-localization profiles showed CDR1 and CDR2 as being the main contributors to variance among IGHV genes, consistent with the importance of these sub-regions in antigen binding. Our results suggest that AID overlapping (WGCW) hotspots alone or in conjunction with Polη (WA/TW) hotspots are key features of evolutionary variation between IGHV genes. [ABSTRACT FROM AUTHOR]

• RNASeq analysis of SRSF1–3 reconstituted cells shows upregulation of SATB2 and UBN1. • Increased SATB2 and UBN1 are enriched in the MAR and promoter regions of IgL gene. • Enhanced occupancy of UBN1, histone H3.3 & SATB2 leads to increased IgL transcription. • Splicing isoform SRSF1–3 regulates alternate splicing pattern of many genes. SRSF1, a member of the SR protein family, is an important splicing factor and regulator of splicing. Multiple splicing isoforms have been reported for this gene. SRSF1–3, a splicing isoform of SRSF1, is necessary for AID-dependent SHM of Ig V genes. However, its precise role in SHM remains enigmatic. Transcriptomic analysis of SRSF1–3 reconstituted cells shows upregulation of transcription factor SATB2 and chromatin regulator UBN1. The increased SATB2 and UBN1 are strikingly enriched in the MAR and promoter regions of the Ig L gene, respectively. Furthermore, UBN1 enrichment at the promoter region was coupled with a hundred-fold enhanced occupancy of the histone variant H3.3 at the Ig L promoter, that is a hallmark of efficient SHM. The enhanced occupancy of SATB2 at the MAR, UBN1 and histone variant H3.3 at the Ig L promoter leads to an increase in Ig L transcription, revealing a role of SRSF1–3 in SHM. Thus, SRSF1–3 is likely involved in the regulation of SHM, via upregulation of a crucial transcription factor SATB2, as well as, by overexpression of a chromatin modulator of Ig genes, UBN1, which further assists in the recruitment of the histone variant H3.3. Furthermore, the splicing isoform SRSF1–3 regulates alternate splicing pattern of splicing isoforms for various crucial genes. The present study provides the first evidence that a splicing isoform of an SR protein can regulate the post-transcriptional processing of RNA in vivo. [ABSTRACT FROM AUTHOR]

• Absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. • Co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase1. • IF and PLA studies corroborated the direct interaction between SRSF1-3 and TOP1. • SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1. Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of Ig V genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1. [ABSTRACT FROM AUTHOR]

The diversity of B cell receptors provides a basis for recognizing numerous pathogens. Antibody repertoire sequencing has revealed relationships between B cell receptor sequences, their diversity, and their function in infection, vaccination, and disease. However, many repertoire datasets have been deposited without annotation or quality control, limiting their utility. To accelerate investigations of B cell immunoglobulin sequence repertoires and to facilitate development of algorithms for their analysis, we constructed a comprehensive public database of curated human B cell immunoglobulin sequence repertoires, cAb-Rep (https://cab-rep.c2b2.columbia.edu), which currently includes 306 immunoglobulin repertoires from 121 human donors, who were healthy, vaccinated, or had autoimmune disease. The database contains a total of 267.9 million V(D)J heavy chain and 72.9 million VJ light chain transcripts. These transcripts are full-length or near full-length, have been annotated with gene origin, antibody isotype, somatic hypermutations, and other biological characteristics, and are stored in FASTA format to facilitate their direct use by most current repertoire-analysis programs. We describe a website to search cAb-Rep for similar antibodies along with methods for analysis of the prevalence of antibodies with specific genetic signatures, for estimation of reproducibility of somatic hypermutation patterns of interest, and for delineating frequencies of somatically introduced N -glycosylation. cAb-Rep should be useful for investigating attributes of B cell sequence repertoires, for understanding characteristics of affinity maturation, and for identifying potential barriers to the elicitation of effective neutralizing antibodies in infection or by vaccination. [ABSTRACT FROM AUTHOR]

• AID preferentially targets non-transcribed strand in a nucleosomal sequence. • The favoured targeting on the top strand is irrespective of UNG inhibition. • Biased targeting persists despite reversing of positioning sequence MP2 to MP2R. • Nucleosome positioning, stability and remodelling contribute to efficient SHM. AID initiates both somatic hypermutation (SHM) and class switch recombination (CSR) in Ig genes. AID-induced mutations are linked with transcription initiation and elongation. Transcription occurs in the context of chromatin and thus RNA PolII and AID need to deal with nucleosomes. Both nucleosome stability and positioning significantly influence the accessibility of AID to Ig genes and the SHM pattern. Interestingly, in the nucleosome, SHM process seems to have a preference for the top strand. To know whether the preferential targeting of SHM to the top strand is due to a post-AID event, we expressed an inhibitor of Uracil DNA glycosylase (UNG), Ugi, into DT40 cells containing the nucleosome positioning sequence (MP2) and compared the SHM pattern. We observed a similar preference to the top strand for the high-affinity nucleosome positioning sequence in UNG inhibited cells. Furthermore, to understand whether the primary sequence of nucleosome sequence is influencing preferential targeting, we introduced two copies of MP2 sequence in the reverse orientation (MP2R) into a variable Ig gene. We observed that the MP2R cells also demonstrated preferential targeting of the non-transcribed strand in nucleosome as compared to the transcribed strand, confirming that in nucleosome sequences AID has better access to Cs on the top strand. The preferential targeting of AID on the top strand suggests that RNA Pol-II stalls while it transcribes the stable nucleosomes, thus giving ample opportunity for the transcribed strand to form R-loops with the nascent RNA, thereby gives limited access to AID on the bottom strand. [ABSTRACT FROM AUTHOR]

The critical role of the CD40/CD40L pathway in B-cell proliferation, immunoglobulin (Ig) isotype switching and germinal center formation has been studied and described extensively in previous literature. Interruption of the CD40/CD40L signal causes hyper-IgM (HIGM) syndrome, which has been classified and recognized as a group of rare inherited immune deficiency disorders. Defects in CD40 and CD40L interactions or in downstream signaling molecules, including activation-induced cytidine deaminase, uracyl-DNA-glycosylase, NF-κB and DNA repair enzymes, result in an increased level of serum IgM and a significantly decreased or absent level of IgA, IgG and IgE that is accompanied by severe recurrent infections and autoimmune diseases. Many genetic defects in HIGM have been identified and, as a result, it is possible for patients to be definitively diagnosed by gene sequencing and to delineate the immunological features of the patients. Modifying the CD40/CD40L signaling pathway may offer the possibility of restoring the normal serum Ab production and curing the immunodeficiency. Hematopoietic stem cell transplantation has achieved a high rate of success using a sibling donor. In addition, successful examples of treating other immunodeficiencies using gene therapy indicated that there was a possibility of eradicating HIGM with this approach. In this review, we summarize the current drugs and a variety of therapeutic approaches for the treatment of the HIGM syndrome by interfering with the defective CD40/CD40L pathway. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID), primarily expressed in activated mature B lymphocytes in germinal centers, is the key factor in adaptive immune response against foreign antigens. AID is responsible for producing high-affinity and high-specificity antibodies against an infectious agent, through the physiological DNA alteration processes of antibody genes by somatic hypermutation (SHM) and class-switch recombination (CSR) and functions by deaminating deoxycytidines (dC) to deoxyuridines (dU), thereby introducing point mutations and double-stranded chromosomal breaks (DSBs). The beneficial physiological role of AID in antibody diversification is outweighed by its detrimental role in the genesis of several chronic immune diseases, under non-physiological conditions. This review offers a comprehensive and better understanding of AID biology and its pathological aspects, as well as addresses the challenges involved in AID-related cancer therapeutics, based on various recent advances and evidence available in the literature till date. In this article, we discuss ways through which our interpretation of AID biology may reflect upon novel clinical insights, which could be successfully translated into designing clinical trials and improving patient prognosis and disease management. [ABSTRACT FROM AUTHOR]

Lymphocytes are endowed with unique and specialized enzymatic mutagenic properties that allow them to diversify their antigen receptors, which are crucial sensors for pathogens and mediators of adaptive immunity. During lymphocyte development, the antigen receptors expressed by B and T lymphocytes are assembled in an antigen-independent fashion by ordered variable gene segment recombinations (V(D)J recombination), which is a highly ordered and regulated process that requires the recombination activating gene products 1 & 2 (RAG1, RAG2). Upon activation by antigen, B lymphocytes undergo additional diversifications of their immunoglobulin B-cell receptors. Enzymatically induced somatic hypermutation (SHM) and immunoglobulin class switch recombination (CSR) improves the affinity for antigen and shape the effector function of the humoral immune response, respectively. The activation-induced cytidine deaminase (AID) enzyme is crucial for both SHM and CSR. These processes have evolved to both utilize as well as evade different DNA repair and DNA damage response pathways. The delicate balance between enzymatic mutagenesis and DNA repair is crucial for effective immune responses and the maintenance of genomic integrity. Not surprisingly, disturbances in this balance are at the basis of lymphoid malignancies by provoking the formation of oncogenic mutations and chromosomal aberrations. In this review, we discuss recent mechanistic insight into the regulation of RAG1/2 and AID expression and activity in lymphocytes and the complex interplay between these mutagenic enzymes and DNA repair and DNA damage response pathways, focusing on the base excision repair and mismatch repair pathways. We discuss how disturbances of this interplay induce genomic instability and contribute to oncogenesis. [ABSTRACT FROM AUTHOR]

The induction of somatic hypermutation (SHM) in various cell lines by activation-induced cytidine deaminase (AID) has been used in protein-directed selection, especially in antibody affinity maturation. Several antibody affinity maturation systems based on mammalian cells have been developed in recent years, i.e., 293T, H1299, Raji and CHO cells. However, the efficiency of in vitro AID-induced hypermutation is low, restricting the application of such systems. In this study, we examined the role of Ig and Ek enhancers in enhancing SHM in the episomal vector pCEP4 that expresses an anti-high mobility group box 1 (HMGB1) full-length antibody. The plasmid containing the two enhancers exhibited two-fold improvement of mutation rate over pCEP4 in an AID expression H1299 cell line (H1299-AID). With the engineered episomal vector, we improved the affinity of this antibody in H1299-AID cells by 20-fold.

ABSTRACT Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation (SHM) for genes of interest in various cells, and several research groups (including ours) have successfully improved antibody affinity in mammalian or chicken cells using AID-induced SHM. These affinity maturation systems are time-consuming and inefficient. In this study, we developed an antibody affinity maturation platform in Chinese hamster ovary (CHO) cells by coupling recombinase-mediated cassette exchange (RMCE) with SHM. Stable CHO cell clones containing a single copy puromycin resistance gene ( PuroR) expression cassette flanked by recombination target sequences (FRT and loxP) being able to highly express a gene of interest placed in the cassette were developed. The PuroR gene was replaced with an antibody gene by RMCE, and the antibody was displayed on the cell surface. Cells displaying antibodies on their membrane were transfected with the AID gene, and mutations of the antibody gene were accumulated by AID-mediated hypermutation during cell proliferation followed by flow cytometric cell sorting for cells bearing antibody mutants with improved affinity. Affinity improvements were detected after only one round of cell sorting and proliferation, mutant clones with 15-fold affinity improvement were isolated within five rounds of maturation (within 2 months). CHO cells are fast growing, stress-resistant and produce antibody with glycosylations suitable for therapy. Our antibody-evolution platform based on CHO cells makes antibody-affinity maturation more efficient and is especially convenient for therapeutic antibody affinity improvement. Biotechnol. Bioeng. 2016;113: 39-51. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Highlights: [•] E2A binds to the UNG2 promoter and represses expression of UNG2. [•] UNG2 preferentially accumulates in regions of the IgH gene containing AID hotspots. [•] Data indicate that this UNG2 accumulation is negatively regulated by E2A as well. [•] Over-expression of E2A can suppress class switch recombination. [•] The results suggest that E2A is regulating the balance between AID and UNG2. [ABSTRACT FROM AUTHOR]

Abstract: Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. [Copyright &y& Elsevier]

Highlights: [•] DNA Pols β and λ do not repair AID-induced lesions during SHM of antibody VDJ genes. [•] The BER pathway is not important for generating mutations at A:T bp. [•] This suggests that most SSBs needed for SHM at A:T bp are made during DNA replication. [•] We propose that during SHM, AID deaminates dCs at the replication fork. [Copyright &y& Elsevier]

Abstract: Activation-induced cytidine deaminase (AID) is a DNA editing protein that plays an essential role in three major events of immunoglobulin (Ig) diversification: somatic hypermutation, class switch recombination and Ig gene conversion. Mutations in the AID gene (AICDA) have been found in patients with autosomal recessive Hyper-IgM (HIGM) syndrome type 2. Here, two 9- and 14-year-old Brazilian sisters, from a consanguineous family, were diagnosed with HIGM2 syndrome. Sequencing analysis of the exons from AICDA revealed that both patients are homozygous for a single C to G transversion in the third position of codon 15, which replaces a conserved Phenylalanine with a Leucine. To our knowledge, this is a new AICDA mutation found in HIGM2 patients. Functional studies confirm that the homologous murine mutation leads to a dysfunctional protein with diminished intrinsic cytidine deaminase activity and is unable to rescue CSR when introduced in Aicda −/−stimulated murine B cells. We briefly discuss the relevance of AICDA mutations found in patients for the biology of this molecule. [Copyright &y& Elsevier]

Activation-induced cytidine deaminase (AID) is expressed in a B cell differentiation stage-specific fashion and is essential for immunoglobulin (Ig) gene class switch DNA recombination (CSR) and somatic hypermutation (SHM). CSR and SHM play a central role in the maturation of antibody and autoantibody responses. AID displays a mutagenic activity by catalyzing targeted deamination of deoxycytidine (dC) residues in DNA resulting in dU:dG mismatches, which are processed into point-mutations in SHM or double-strand breaks (DSBs) in CSR. Although AID specifically targets the Ig gene loci ( IgH, Igκ and Igλ), it can also home into a wide array of non-Ig genes in B-and non-B-cell backgrounds. Aberrant expression of AID is associated with multiple diseases such as allergy, inflammation, autoimmunity and cancer. In autoimmune systemic lupus erythematosus, dysregulated AID expression underpins increased CSR, SHM and autoantibody production. As a potent mutator, AID is under stringent transcriptional, post-transcriptional and post-translational regulation. AID is also regulated in its targeting and enzymatic function. In resting naïve or memory B cells, AID transcripts and protein are undetectable. These, however, are readily and significantly up-regulated in B cells induced to undergo CSR and/or SHM. Transcription factors, such as HoxC4 and NF-κB, which are up-regulated in a B cell lineage-and/or differentiation stage-specific manner, regulate the induction of AID. HoxC4 induces AID expression by directly binding to the AID gene promoter through an evolutionarily conserved 5′-ATTT-3′ motif. HoxC4 is induced by the same stimuli that induce AID and CSR. It is further up-regulated by estrogen through three estrogen responsive elements in its promoter region. The targeting of AID to switch (S) regions is mediated by 14-3-3 adaptor proteins, which specifically bind to 5′-AGCT-3′ repeats that are exist at high frequency in S region cores. Like HoxC4, 14-3-3 adaptors are induced by the same stimuli that induce AID. These include 'primary' inducing stimuli, that is, those that play a major role in inducing AID, i.e., engagement of CD40 by CD154, engagement of Toll-like receptors (TLRs) by microbial-associated molecular patterns (MAMPs) and cross-linking of the BCR, as synergized by 'secondary' inducing stimuli, that is, those that synergize for AID induction and specify CSR to different isotypes, i.e., switch-directing cytokines IL-4, TGF-β or IFN-γ. In this review, we focus on the multi-levels regulation of AID expression and activity. We also discuss the dysregulation or misexpression of AID in autoimmunity and tumorigenesis. [ABSTRACT FROM AUTHOR]

Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WR C motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM. [ABSTRACT FROM AUTHOR]

Activation-induced deaminase (AID) only deaminates cytosine within single-stranded DNA. Transcription is known to increase AID deamination on duplex DNA substrates during transcription. Using a purified T7 RNA polymerase transcription system, we recently found that AID deamination of a duplex DNA substrate is reduced if RNase A is added during transcription. This finding prompted us to consider that the mRNA tail may contribute to AID action at the nearby transcribed strand (TS) or non-transcribed strand (NTS) of DNA, which are transiently single-stranded in the wake of RNA polymerase movement. Here, we used a purified system to test whether a single-stranded oligonucleotide (oligo) consisting of RNA in the 5′ portion and DNA in the 3′ portion (i.e., 5′RNA-DNA3′, also termed an RNA-DNA fusion substrate) could be deaminated equally efficiently as the same sequence when it is entirely DNA. We found that AID acts on the RNA-DNA fusion substrate and the DNA-only substrate with similar efficiency. Based on this finding and our recent observation on the importance of the mRNA tail, we propose a model in which the proximity and length of the mRNA tail provide a critical site for AID loading to permit a high local collision frequency with the NTS and TS in the transient wake of the RNA polymerase. When the mRNA tail is not present, we know that AID action drops to levels equivalent to when there is no transcription at all. This mRNA tether model explains several local and global features of Ig somatic hypermutation and Ig class switch recombination, while integrating structural and functional features of AID. • AID deaminates DNA in a fusion substrate with RNA in the 5′ and DNA in the 3′ end. • AID action on DNA-only versus RNA-DNA substrates of equal length are the same. • We propose a model where the mRNA tail recruits AID during transcription. • This model would explain key aspects of Ig somatic hypermutation and class switch. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/faslpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/faslpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/faslpr/lpr B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/faslpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/faslpr/lpr mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) θ, pol η and pol ζ, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/faslpr/lpr mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions. [ABSTRACT FROM AUTHOR]

Abstract: The DNA sequence data of the somatic hypermutation (SHM) field published since 1984 has been critically reviewed. The analysis has revealed three strand biased mutation signatures. The first concerns the mutations generated at G:C base pairs in mice genetically deficient in uracil-DNA glycosylase and MSH2–MSH6-mediated mismatch repair. Such mice display the AID deaminase footprint and here C mutations exceed G mutations at least 1.5-fold. This supports earlier and more recent studies claiming that dC-to-dU deaminations occur preferentially in the single stranded DNA regions of the displaced nontranscribed strand (NTS) during transcription. The second concerns the signature generated in immunised mice where G mutations exceed C mutations by at least 1.7-fold. This is a newly identified strand bias which has previously gone undetected. It is consistent with the polynucleotide polymerisation signature of RNA polymerase II copying the template DNA strand carrying AID-mediated lesions generated at C bases, viz. uracils and abasic sites. A reverse transcription step would then need to intervene to fix the mutation pattern in DNA. The third concerns the long recognised strand biased signature generated in normal aged or actively immunised mice whereby A mutations exceed T mutations by two- to three-fold. It is argued that this pattern is best understood as a combination of adenosine-to-inosine (A-to-I) RNA editing followed by a reverse transcription step fixing the A-to-G, as well as A-to-T and A-to-C, as strand biased mutation signatures in DNA. The reasons why the AID-linked RNA polymerase II mutation signature had previously gone undetected are discussed with regard to limitations of standard PCR-based SHM assay techniques. It is concluded that the most economical SHM mechanism involves both DNA and RNA deaminations coupled to a reverse transcription process, most likely involving DNA polymerase η acting in its reverse transcriptase mode. Experimental approaches to differentiate this RNA-based model from the standard DNA deamination model are discussed. [Copyright &y& Elsevier]

Abstract: During the immune response, the generation of memory B lymphocytes in germinal centers involves affinity maturation of the cells’ antigen receptors, based on somatic hypermutation of receptor genes and antigen-driven selection of the resulting mutants. Affinity maturation is vital for immune protection, and is the basis of humoral immune learning and memory. Lineage trees of somatically hypermutated immunoglobulin genes often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation in germinal centers. Here, we derive the quantitative relationships between parameters characterizing affinity maturation dynamics (proliferation, differentiation and mutation rates, initial affinity of the Ig to the antigen, and selection thresholds) and the mathematical properties of lineage trees, using a computer simulation which combines mathematical models for all mature B cell populations, stochastic models of hypermutation and selection, lineage tree generation and measurement of graphical tree characteristics. We identified seven key lineage tree properties, and found correlations of these with initial clone affinity and with the selection threshold. These two parameters were found to be the main factors affecting lineage tree shapes in both primary and secondary response trees. The results also confirm that recycling from centrocytes back to centroblasts is highly likely. [Copyright &y& Elsevier]

Immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are critical for the maturation of the antibody response. These processes endow antibodies with increased antigenbinding affinity and acquisition of new biological effector functions, thereby underlying the generation of memory B cells and plasma cells. They are dependent on the generation of specific DNA lesions and the intervention of activation-induced cytidine deaminase as well as newly identified translesion DNA polymerases, which are expressed in germinal center B cells. DNA lesions include mismatches, abasic sites, nicks, single-strand breaks, and doublestrand breaks (DSBs). DSBs in the switch (S) region DNA are critical for CSR, but they also occur in V(D)J regions and possibly contribute to the events that lead to SHM. The nature of the DSBs in the Ig locus, their generation, and the repair processes that they trigger and that are responsible for their regulation remain poorly understood. Aberrant regulation of these events can result in chromosomal breaks and translocations, which are significant steps in B-cell neoplastic transformation. [ABSTRACT FROM AUTHOR]

Immunoglobulin A is the main element of the humoral immune response that has been selected through evolution, together with innate mucosal defences, to provide protection against microbial antigens at mucosal surfaces. IgA responses are initiated in organized inductive structures, such as Peyer’s patches and nasal-associated lymphoid tissues, as well as diffuse effector tissues, such as gut lamina propria and nasal mucosa. Hypermutated secretory IgAs play a critical role in regulating the composition of the intestinal microflora. Dysregulation of gut homeostasis in IgA-deficient gut causes a continuous activation of the immune cells and induces inflammatory processes leading to lymphoneogenesis. Recent advances in this field include new insights into the role of IgA in the maintenance of gut homeostasis and the proposal of an updated model for the induction of IgA responses in the gut. [Copyright &y& Elsevier]

Somatic hypermutation and class switch recombination cause genetic alterations in immunoglobulin (Ig) genes, which underlie the generation of the secondary antibody repertoire in B lymphocytes. Both processes require activation-induced cytidine deaminase (AID), whose mechanism of action in not yet known in detail, but which mediates the accumulation of point mutations in the Ig locus. This highly mutagenic process must be tightly controlled, and multiple levels of regulation might exist. Recent experiments show that AID deaminates deoxycytidine to deoxyuridine in single-stranded DNA. This mutagenic event is targeted to actively transcribed sequences, and the specificity of deamination might be related to the chromatin structure of the transcription complex. [Copyright &y& Elsevier]

The generation, development, maturation and selection of mammalian B lymphocytes is a complex process that is initiated in the embryo and proceeds throughout life to provide the organism an essential part of the immune system it requires to cope with pathogens. Transcriptional regulation of this highly complex series of events is a major control mechanism, although control is also exerted on all other layers, including splicing, translation and protein stability. This review summarizes our current understanding of transcriptional control of the well-studied murine B cell development, which bears strong similarity to its human counterpart. Animal and cell models with loss of function (gene “knock outs”) or gain of function (often transgenes) have significantly contributed to our knowledge about the role of specific transcription factors during B lymphopoiesis. In particular, a large number of different transcriptional regulators have been linked to distinct stages of the life of B lymphocytes such as: differentiation in the bone marrow, migration to the peripheral organs and antigen-induced activation. [Copyright &y& Elsevier]

Bcl-2 translocations in follicular lymphomas (FL) are often associated with downstream immunoglobulin class switch recombination (CSR) on the translocated allele. We studied cell lines with different translocations into the IgH locus to gauge any common features associated with downstream CSR events. CSR associated with chromosomal rearrangements was observed in cells (RL) with translocations similar to those frequently observed in FL (bcl-2-JH), and such CSR was also seen with a myc (near exon 1)-JH5 intron translocation (MC116), but not for far 5′-myc-JH5 intron (P3HR-1) or myc-Sμ translocations (Ramos). Both MC116 and P3HR-1 myc translocations showed evidence for an origin from somatic hypermutation. Therefore, the association of JH translocations with CSR on the translocated allele is unlikely to be linked with specific translocation mechanisms, and the P3HR-1 configuration indicated that the downstream class switching is not a necessary consequence of (or precondition for) such a translocation event. MC116 and RL, but not P3HR-1 cells, showed constitutive transcription through the translocated IgH alleles, suggesting that transcription through this region or the processing of such transcripts may promote CSR. However, while CSR events clearly occurred in the precursors of MC116 and RL, neither cell line could undergo complete class switching. [Copyright &y& Elsevier]

The immune system makes use of two distinct mechanisms to mount an efficient response against almost every foreign macromolecular substance. First, antibodies with their robust immunoglobulin domain architecture provide a rigid scaffold to support six hypervariable loops, capable of forming highly diverse binding sites. Second, an efficient genetic mechanism has evolved to create sequence diversity at the somatic level in a step-wise process, whereby random recombination of an inherited set of gene segments is followed by hypermutation events. Insight into the corresponding molecular mechanisms is developing rapidly and enables adaptation of the emerging principles to the creation of artificial binding proteins in vitro, using the techniques of combinatorial biotechnology. Thus, novel types of receptor molecules have been constructed from alternative scaffolds, including α-helical bundle and β-barrel proteins. These may provide superior tools for the recognition, targeting or separation of a wide range of biomolecular structures or substances in biological research, technology, and even medicine. [Copyright &y& Elsevier]