TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate.

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.

Immune Monitoring of Trans-endothelial Transport by Kidney-Resident Macrophages.

Small immune complexes cause type III hypersensitivity reactions that frequently result in tissue injury. The responsible mechanisms, however, remain unclear and differ depending on target organs. Here, we identify a kidney-specific anatomical and functional unit, formed by resident macrophages and peritubular capillary endothelial cells, which monitors the transport of proteins and particles ranging from 20 to 700 kDa or 10 to 200 nm into the kidney interstitium. Kidney-resident macrophages detect and scavenge circulating immune complexes "pumped" into the interstitium via trans-endothelial transport and trigger a FcγRIV-dependent inflammatory response and the recruitment of monocytes and neutrophils. In addition, FcγRIV and TLR pathways synergistically "super-activate" kidney macrophages when immune complexes contain a nucleic acid. These data identify a physiological function of tissue-resident kidney macrophages and a basic mechanism by which they initiate the inflammatory response to small immune complexes in the kidney.

A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization.

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

MRI Is a DNA Damage Response Adaptor during Classical Non-homologous End Joining.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Outflanking immunodominance to target subdominant broadly neutralizing epitopes.

A major obstacle to vaccination against antigenically variable viruses is skewing of antibody responses to variable immunodominant epitopes. For influenza virus hemagglutinin (HA), the immunodominance of the variable head impairs responses to the highly conserved stem. Here, we show that head immunodominance depends on the physical attachment of head to stem. Stem immunogenicity is enhanced by immunizing with stem-only constructs or by increasing local HA concentration in the draining lymph node. Surprisingly, coimmunization of full-length HA and stem alters stem-antibody class switching. Our findings delineate strategies for overcoming immunodominance, with important implications for human vaccination.

Cutting Edge: ATM Influences Germinal Center Integrity.

The DNA damage response protein ATM has long been known to influence class switch recombination in ex vivo-cultured B cells. However, an assessment of B cell-intrinsic requirement of ATM in humoral responses in vivo was confounded by the fact that its germline deletion affects T cell function, and B:T cell interactions are critical for in vivo immune responses. In this study, we demonstrate that B cell-specific deletion of ATM in mice leads to reduction in germinal center (GC) frequency and size in response to immunization. We find that loss of ATM induces apoptosis of GC B cells, likely due to unresolved DNA lesions in cells attempting to undergo class-switch recombination. Accordingly, suboptimal GC responses in ATM-deficient animals are characterized by decreased titers of class-switched Abs and decreased rates of somatic hypermutation. These results unmask the critical B cell-intrinsic role of ATM in maintaining an optimal GC response following immunization.

BRCT-domain protein BRIT1 influences class switch recombination.

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

A transcriptional serenAID: the role of noncoding RNAs in class switch recombination.

During an immune response, activated B cells may undergo class switch recombination (CSR), a molecular rearrangement that allows B cells to switch from expressing IgM and IgD to a secondary antibody heavy chain isotype such as IgG, IgA or IgE. Secondary antibody isotypes provide the adaptive immune system with distinct effector functions to optimally combat various pathogens. CSR occurs between repetitive DNA elements within the immunoglobulin heavy chain (Igh) locus, termed switch (S) regions and requires the DNA-modifying enzyme activation-induced cytidine deaminase (AID). AID-mediated DNA deamination within S regions initiates the formation of DNA double-strand breaks, which serve as biochemical beacons for downstream DNA repair pathways that coordinate the ligation of DNA breaks. Myriad factors contribute to optimal AID targeting; however, many of these factors also localize to genomic regions outside of the Igh locus. Thus, a current challenge is to explain the specific targeting of AID to the Igh locus. Recent studies have implicated noncoding RNAs in CSR, suggesting a provocative mechanism that incorporates Igh-specific factors to enable precise AID targeting. Here, we chronologically recount the rich history of noncoding RNAs functioning in CSR to provide a comprehensive context for recent and future discoveries. We present a model for the RNA-guided targeting of AID that attempts to integrate historical and recent findings, and highlight potential caveats. Lastly, we discuss testable hypotheses ripe for current experimentation, and explore promising ideas for future investigations.

IL-21 Shapes the B Cell Response in a Context-Dependent Manner.

The cytokine interleukin-21 (IL-21) is a pivotal T cell-derived signal crucial for germinal center (GC) responses, but the precise mechanisms by which IL-21 influences B cell function remain elusive. Here, we investigated the B cell-intrinsic role of IL-21 signaling by employing a novel IL-21 receptor ( Il21r ) conditional knock-out mouse model and ex vivo culture systems and uncovered a surprising duality of IL-21 signaling in B cells. While IL-21 stimulation of naïve B cells led to Bim-dependent apoptosis, it promoted robust proliferation of pre-activated B cells, particularly class-switched IgG1 + B cells ex vivo . Consistent with this, B cell-specific deletion of Il21r led to a severe defect in IgG1 responses in vivo following immunization. Intriguingly, Il21r -deleted B cells are significantly impaired in their ability to transition from a pre-GC to a GC state following immunization. Although Il21r -deficiency did not affect the proportion of IgG1 + B cells among GC B cells, it greatly diminished the proportion of IgG1 + B cells among the plasmablast/plasma cell population. Collectively, our data suggest that IL-21 serves as a critical regulator of B cell fates, influencing B cell apoptosis and proliferation in a context-dependent manner.

Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia.

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P < 0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

Exportin Crm1 is important for Swi6 nuclear shuttling and MBF transcription activation in Saccharomyces cerevisiae.

BACKGROUND: Swi6 acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. This study investigates how nucleocytoplasmic transport by multiple transport factors may influence specific Swi6 activities. RESULTS: Here we show that the exportin Crm1 is important for Swi6 nuclear export and activity. Loss of a putative Crm1 NES or inhibition of Crm1 activity results in changes in nucleocytoplasmic Swi6 localization. Alteration of the Crm1 NES in Swi6 results in decreased MBF-mediated gene expression, but does not affect SBF reporter expression, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the putative Crm1 NES in Swi6 results in cells that are larger than wild type, and this increase in cell size is exacerbated by deletion of Msn5. CONCLUSIONS: These data provide evidence that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal. We identify a putative nuclear export signal for Crm1 within Swi6, and observe that export by Crm1 or Msn5 independently influences Swi6-regulated expression of a different subset of Swi6-controlled genes. These findings provide new insights into the complex regulation of Swi6 transcription activation activity and the role of nucleocytoplasmic shuttling in regulated gene expression.

Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells.

Androgens have been used in the treatment of bone marrow failure syndromes without a clear understanding of their mechanism of action. Blood counts of patients with dyskeratosis congenita or aplastic anemia with mutations in telomerase genes can improve with androgen therapy. Here we observed that exposure in vitro of normal peripheral blood lymphocytes and human bone marrow-derived CD34(+) cells to androgens increased telomerase activity, coincident with higher TERT mRNA levels. Cells from patients who were heterozygous for telomerase mutations had low baseline telomerase activity, which was restored to normal levels by exposure to androgens. Estradiol had an effect similar to androgens on TERT gene expression and telomerase enzymatic activity. Tamoxifen abolished the effects of both estradiol and androgens on telomerase function, and letrozole, an aromatase inhibitor, blocked androgen effects on telomerase activity. Conversely, flutamide, an androgen receptor antagonist, did not affect androgen stimulation of telomerase. Down-regulation by siRNA of estrogen receptor-alpha (ER alpha), but not ER beta, inhibited estrogen-stimulated telomerase function. Our results provide a mechanism for androgen therapy in bone marrow failure: androgens appear to regulate telomerase expression and activity mainly by aromatization and through ER alpha. These findings have potential implications for the choice of current androgenic compounds and the development of future agents for clinical use.

Assembly of a spatial circuit of T-bet-expressing T and B lymphocytes is required for antiviral humoral immunity.

Effective antiviral immunity requires generation of T and B lymphocytes expressing the transcription factor T-bet, a regulator of type 1 inflammatory responses. Using T-bet expression as an endogenous marker for cells participating in a type 1 response, we report coordinated interactions of T-bet-expressing T and B lymphocytes on the basis of their dynamic colocalization at the T cell zone and B follicle boundary (T-B boundary) and germinal centers (GCs) during lung influenza infection. We demonstrate that the assembly of this circuit takes place in distinct anatomical niches within the draining lymph node, guided by CXCR3 that enables positioning of TH1 cells at the T-B boundary. The encounter of B and TH1 cells at the T-B boundary enables IFN-γ produced by the latter to induce IgG2c class switching. Within GCs, T-bet+ TFH cells represent a specialized stable sublineage required for GC growth but dispensable for IgG2c class switching. Our studies show that during respiratory viral infection, T-bet-expressing T and B lymphocytes form a circuit assembled in a spatiotemporally controlled manner that acts as a functional unit enabling a robust and coherent humoral response tailored for optimal antiviral immunity.

Temporal dynamics of persistent germinal centers and memory B cell differentiation following respiratory virus infection.

Following infection or immunization, memory B cells (MBCs) and long-lived plasma cells provide humoral immunity that can last for decades. Most principles of MBC biology have been determined with hapten-protein carrier models or fluorescent protein immunizations. Here, we examine the temporal dynamics of the germinal center (GC) B cell and MBC response following mouse influenza A virus infection. We find that antiviral B cell responses within the lung-draining mediastinal lymph node (mLN) and the spleen are distinct in regard to duration, enrichment for antigen-binding cells, and class switching dynamics. While splenic GCs dissolve after 6 weeks post-infection, mLN hemagglutinin-specific (HA+) GCs can persist for 22 weeks. Persistent GCs continuously differentiate MBCs, with "peak" and "late" GCs contributing equal numbers of HA+ MBCs to the long-lived compartment. Our findings highlight critical aspects of persistent GC responses and MBC differentiation following respiratory virus infection with direct implications for developing effective vaccination strategies.

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

Distinct Requirements of CHD4 during B Cell Development and Antibody Response.

The immunoglobulin heavy chain (Igh) locus features a dynamic chromatin landscape to promote class switch recombination (CSR), yet the mechanisms that regulate this landscape remain poorly understood. CHD4, a component of the chromatin remodeling NuRD complex, directly binds H3K9me3, an epigenetic mark present at the Igh locus during CSR. We find that CHD4 is essential for early B cell development but is dispensable for the homeostatic maintenance of mature, naive B cells. However, loss of CHD4 in mature B cells impairs CSR because of suboptimal targeting of AID to the Igh locus. Additionally, we find that CHD4 represses p53 expression to promote B cell proliferation. This work reveals distinct roles for CHD4 in B cell development and CSR and links the H3K9me3 epigenetic mark with AID recruitment to the Igh locus.

RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin.

Heterochromatin formed by the SUV39 histone methyltransferases represses transcription from repetitive DNA sequences and ensures genomic stability. How SUV39 enzymes localize to their target genomic loci remains unclear. Here, we demonstrate that chromatin-associated RNA contributes to the stable association of SUV39H1 with constitutive heterochromatin in human cells. We find that RNA associated with mitotic chromosomes is concentrated at pericentric heterochromatin, and is encoded, in part, by repetitive α-satellite sequences, which are retained in cis at their transcription sites. Purified SUV39H1 directly binds nucleic acids through its chromodomain; and in cells, SUV39H1 associates with α-satellite RNA transcripts. Furthermore, nucleic acid binding mutants destabilize the association of SUV39H1 with chromatin in mitotic and interphase cells - effects that can be recapitulated by RNase treatment or RNA polymerase inhibition - and cause defects in heterochromatin function. Collectively, our findings uncover a previously unrealized function for chromatin-associated RNA in regulating constitutive heterochromatin in human cells.

The aryl hydrocarbon receptor controls cell-fate decisions in B cells.

Generation of cellular heterogeneity is an essential feature of the adaptive immune system. This is best exemplified during humoral immune response when an expanding B cell clone assumes multiple cell fates, including class-switched B cells, antibody-secreting plasma cells, and memory B cells. Although each cell type is essential for immunity, their generation must be exquisitely controlled because a class-switched B cell cannot revert back to the parent isotype, and a terminally differentiated plasma cell cannot contribute to the memory pool. In this study, we show that an environmental sensor, the aryl hydrocarbon receptor (AhR) is highly induced upon B cell activation and serves a critical role in regulating activation-induced cell fate outcomes. We find that AhR negatively regulates class-switch recombination ex vivo by altering activation-induced cytidine deaminase expression. We further demonstrate that AhR suppresses class switching in vivo after influenza virus infection and immunization with model antigens. In addition, by regulating Blimp-1 expression via Bach2, AhR represses differentiation of B cells into plasmablasts ex vivo and antibody-secreting plasma cells in vivo. These experiments suggest that AhR serves as a molecular rheostat in B cells to brake the effector response, possibly to facilitate optimal recall responses. Thus, AhR might represent a novel molecular target for manipulation of B cell responses during vaccination.