Gene Editing of Primary Rhesus Macaque B Cells.

B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.

Distinct gene expression by expanded clones of quiescent memory CD4+ T cells harboring intact latent HIV-1 proviruses.

Antiretroviral therapy controls, but does not cure, HIV-1 infection due to a reservoir of rare CD4+ T cells harboring latent proviruses. Little is known about the transcriptional program of latent cells. Here, we report a strategy to enrich clones of latent cells carrying intact, replication-competent HIV-1 proviruses from blood based on their expression of unique T cell receptors. Latent cell enrichment enabled single-cell transcriptomic analysis of 1,050 CD4+ T cells belonging to expanded clones harboring intact HIV-1 proviruses from 6 different individuals. The analysis reveals that most of these cells are T effector memory cells that are enriched for expression of HLA-DR, HLA-DP, CD74, CCL5, granzymes A and K, cystatin F, LYAR, and DUSP2. We conclude that expanded clones of latent cells carrying intact HIV-1 proviruses persist preferentially in a distinct CD4+ T cell population, opening possibilities for eradication.

Evolution of antibody immunity to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations.

Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.

Combination anti-HIV-1 antibody therapy is associated with increased virus-specific T cell immunity.

Combination antiretroviral therapy (ART) is highly effective in controlling human immunodeficiency virus (HIV)-1 but requires lifelong medication due to the existence of a latent viral reservoir1,2. Potent broadly neutralizing antibodies (bNAbs) represent a potential alternative or adjuvant to ART. In addition to suppressing viremia, bNAbs may have T cell immunomodulatory effects as seen for other forms of immunotherapy3. However, this has not been established in individuals who are infected with HIV-1. Here, we document increased HIV-1 Gag-specific CD8+ T cell responses in the peripheral blood of all nine study participants who were infected with HIV-1 with suppressed blood viremia, while receiving bNAb therapy during ART interruption4. Increased CD4+ T cell responses were detected in eight individuals. The increased T cell responses were due both to newly detectable reactivity to HIV-1 Gag epitopes and the expansion of pre-existing measurable responses. These data demonstrate that bNAb therapy during ART interruption is associated with enhanced HIV-1-specific T cell responses. Whether these augmented T cell responses can contribute to bNAb-mediated viral control remains to be determined.

Plasmodium Infection Promotes Genomic Instability and AID-Dependent B Cell Lymphoma.

Chronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt's lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells. PAPERCLIP.

Epigenetic targeting of activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating cytosine residues in immunoglobulin genes (Igh, Igκ, and Igλ). At a lower frequency, AID also causes collateral DNA damage at non-Ig loci, including genes that are rearranged or mutated in B-cell lymphoma. Precisely how AID is recruited to these off-target sites is not entirely understood. To gain further insight into how AID selects its targets, we compared AID-mediated translocations in two different cell types, B cells and mouse embryonic fibroblasts (MEFs). AID targets a distinct set of hotspots in the two cell types. In both cases, hotspots are concentrated in highly transcribed but stalled genes. However, transcription alone is insufficient to recruit AID activity. Comparison of genes similarly transcribed in B cells and MEFs but targeted in only one of the two cell types reveals a common set of epigenetic features associated with AID recruitment in both cells. AID target genes are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac) and marks of active transcription (such as H3K36me3) in both fibroblasts and B cells, indicating that these features are universal mediators of AID recruitment.

B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity.

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.

Identification of chromosomal translocation hotspots via scan statistics.

MOTIVATION: The detection of genomic regions unusually rich in a given pattern is an important undertaking in the analysis of next-generation sequencing data. Recent studies of chromosomal translocations in activated B lymphocytes have identified regions that are frequently translocated to c-myc oncogene. A quantitative method for the identification of translocation hotspots was crucial to this study. Here we improve this analysis by using a simple probabilistic model and the framework provided by scan statistics to define the number and location of translocation breakpoint hotspots. A key feature of our method is that it provides a global chromosome-wide nominal control level to clustering, as opposed to previous methods based on local criteria. While being motivated by a specific application, the detection of unusual clusters is a widespread problem in bioinformatics. We expect our method to be useful in the analysis of data from other experimental approaches such as of ChIP-seq and 4C-seq. RESULTS: The analysis of translocations from B lymphocytes with the method described here reveals the presence of longer hotspots when compared with those defined previously. Further, we show that the hotspot size changes substantially in the absence of DNA repair protein 53BP1. When 53BP1 deficiency is combined with overexpression of activation-induced cytidine deaminase, the hotspot length increases even further. These changes are not detected by previous methods that use local significance criteria for clustering. Our method is also able to identify several exclusive translocation hotspots located in genes of known tumor supressors. AVAILABILITY AND IMPLEMENTATION: The detection of translocation hotspots is done with hot_scan, a program implemented in R and Perl. Source code and documentation are freely available for download at https://github.com/itojal/hot_scan.

Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching.

DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to the loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether 53BP1 does so directly is not known. Here, we identify Rap1-interacting factor 1 (Rif1) as an ATM (ataxia-telangiectasia mutated) phosphorylation-dependent interactor of 53BP1 and show that absence of Rif1 results in 5'-3' DNA-end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G(1) and S phases of the cell cycle, interferes with class switch recombination in B lymphocytes, and leads to accumulation of chromosome DSBs.

53BP1 alters the landscape of DNA rearrangements and suppresses AID-induced B cell lymphoma.

Deficiencies in factors that regulate the DNA damage response enhance the incidence of malignancy by destabilizing the genome. However, the precise influence of the DNA damage response on regulation of cancer-associated rearrangements is not well defined. Here we examine the genome-wide impact of tumor protein P53-binding protein 1 (53BP1) deficiency in lymphoma and translocation. While both activation-induced cytidine deaminase (AID) and 53BP1 have been associated with cancer in humans, neither AID overexpression nor loss of 53BP1 is sufficient to produce malignancy. However, the combination of 53BP1 deficiency and AID deregulation results in B cell lymphoma. Deep sequencing of the genome of 53BP1(-/-) cancer cells and translocation capture sequencing (TC-Seq) of primary 53BP1(-/-) B cells revealed that their chromosomal rearrangements differ from those found in wild-type cells in that they show increased DNA end resection. Moreover, loss of 53BP1 alters the translocatome by increasing rearrangements to intergenic regions.

DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes.

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

Robust chromosomal DNA repair via alternative end-joining in the absence of X-ray repair cross-complementing protein 1 (XRCC1).

Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI-induced DSBs in XRCC4-deficient pro-B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.

Translocation capture sequencing: a method for high throughput mapping of chromosomal rearrangements.

Chromosomal translocations require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are involved in producing leukemias, lymphomas and sarcomas. Translocations are frequent, clonal and recurrent in mature B cell lymphomas, which bear a particularly high DNA damage burden by virtue of activation-induced cytidine deaminase (AID) expression. Despite the ubiquity of genomic rearrangements, the forces that underlie their genesis are not well understood. Here, we provide a detailed description of a new method for studying these events, translocation capture sequencing (TC-Seq). TC-Seq provides the means to document chromosomal rearrangements genome-wide in primary cells, and to discover recombination hotspots. Demonstrating its effectiveness, we successfully estimate the frequency of c-myc/IgH translocations in primary B cells, and identify hotspots of AID-mediated recombination. Furthermore, TC-Seq can be adapted to generate genome-wide rearrangement maps in any cell type and under any condition.

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.

Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.

Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.

Human anti-HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness.

The identification and characterization of conserved epitopes on the HIV-1 viral spike that are immunogenic in humans and targeted by neutralizing antibodies is an important step in vaccine design. Antibody cloning experiments revealed that 32% of all HIV-neutralizing antibodies expressed by the memory B cells in patients with high titers of broadly neutralizing antibodies recognize one or more "core" epitopes that were not defined. Here, we show that anti-core antibodies recognize a single conserved epitope on the gp120 subunit. Amino acids D474, M475, R476, which are essential for anti-core antibody binding, form an immunodominant triad at the outer domain/inner domain junction of gp120. The mutation of these residues to alanine impairs viral fusion and fitness. Thus, the core epitope, a frequent target of anti-HIV-neutralizing antibodies, including the broadly neutralizing antibody HJ16, is conserved and indispensible for viral infectivity. We conclude that the core epitope should be considered as a target for vaccine design.

PTIP promotes chromatin changes critical for immunoglobulin class switch recombination.

Programmed genetic rearrangements in lymphocytes require transcription at antigen receptor genes to promote accessibility for initiating double-strand break (DSB) formation critical for DNA recombination and repair. Here, we showed that activated B cells deficient in the PTIP component of the MLL3 (mixed-lineage leukemia 3)-MLL4 complex display impaired trimethylation of histone 3 at lysine 4 (H3K4me3) and transcription initiation of downstream switch regions at the immunoglobulin heavy-chain (Igh) locus, leading to defective immunoglobulin class switching. We also showed that PTIP accumulation at DSBs contributes to class switch recombination (CSR) and genome stability independently of Igh switch transcription. These results demonstrate that PTIP promotes specific chromatin changes that control the accessibility of the Igh locus to CSR and suggest a nonredundant role for the MLL3-MLL4 complex in altering antibody effector function.

Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8.

53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1's interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase-dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain-associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX(-/-) mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8(-/-) males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage-inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.

Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain (IgH) constant region exons. CSR is completed by joining a DSB in the donor S mu to a DSB in a downstream acceptor S region (e.g., S gamma1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into S mu during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both S mu and S gamma1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.