Extra centrosomes delay DNA damage-driven tumorigenesis.

Deregulated centrosome numbers are frequently found in human cancer and can promote malignancies in model organisms. Current research aims to clarify if extra centrosomes are cause or consequence of malignant transformation, and if their biogenesis can be targeted for therapy. Here, we show that oncogene-driven blood cancer is inert to genetic manipulation of centrosome numbers, whereas the formation of DNA damage-induced malignancies is delayed. We provide first evidence that this unexpected phenomenon is connected to extra centrosomes eliciting a pro-death signal engaging the apoptotic machinery. Apoptosis induction requires the PIDDosome multi-protein complex, as it can be abrogated by loss of any of its three components, Caspase-2, Raidd/Cradd, or Pidd1. BCL2 overexpression equally blocks cell death, documenting for the first time induction of mitochondrial apoptosis downstream of extra centrosomes. Our findings demonstrate context-dependent effects of centrosome amplification during transformation and ask to adjust current belief that extra centrosomes are intrinsically pro-tumorigenic.

Age-related neuroimmune signatures in dorsal root ganglia of a Fabry disease mouse model.

Pain in Fabry disease (FD) is generally accepted to result from neuronal damage in the peripheral nervous system as a consequence of excess lipid storage caused by alpha-galactosidase A (α-Gal A) deficiency. Signatures of pain arising from nerve injuries are generally associated with changes of number, location and phenotypes of immune cells within dorsal root ganglia (DRG). However, the neuroimmune processes in the DRG linked to accumulating glycosphingolipids in Fabry disease are insufficiently understood.Therefore, using indirect immune fluorescence microscopy, transmigration assays and FACS together with transcriptomic signatures associated with immune processes, we assessed age-dependent neuroimmune alterations in DRG obtained from mice with a global depletion of α-Gal A as a valid mouse model for FD. Macrophage numbers in the DRG of FD mice were unaltered, and BV-2 cells as a model for monocytic cells did not show augmented migratory reactions to glycosphingolipids exposure suggesting that these do not act as chemoattractants in FD. However, we found pronounced alterations of lysosomal signatures in sensory neurons and of macrophage morphology and phenotypes in FD DRG. Macrophages exhibited reduced morphological complexity indicated by a smaller number of ramifications and more rounded shape, which were age dependent and indicative of premature monocytic aging together with upregulated expression of markers CD68 and CD163.In our FD mouse model, the observed phenotypic changes in myeloid cell populations of the DRG suggest enhanced phagocytic and unaltered proliferative capacity of macrophages as compared to wildtype control mice. We suggest that macrophages may participate in FD pathogenesis and targeting macrophages at an early stage of FD may offer new treatment options other than enzyme replacement therapy.

miR-142 favors naïve B cell residence in peripheral lymph nodes.

B lymphocyte development proceeds through a well-ordered sequence of steps, leading to the formation of a sizeable mature B population recognizing a diversity of antigens. These latter cells are ultimately responsible for the production of antibodies upon immune challenges. The detection of threats to the organism is facilitated by the ability of naïve follicular B cells, the main subset of mature B cells in mice, to circulate between lymphoid tissues in search of their cognate antigens. miRNA-mediated fine-tuning of mRNA stability and translation participates in the optimal expression of genetic programs. This regulatory mechanism has been shown to contribute to B cell biology, although the role of individual miRNAs remains understudied. Here, we selectively inactivated the miR-142 locus in B cells. As a consequence, the mature B compartment was visibly perturbed, in agreement with work in miR-142 knockout mice. However, our strategy allowed us to identify roles for the miR-142 locus in B cell physiology obscured by the complexity of the immune phenotype in the null mutant mice. Thus, these miRNAs are necessary for the proper formation of the pre-B cell compartment during development. More remarkably, naïve follicular B cells demonstrated altered migratory properties upon conditional inactivation of the miR-142 locus. The latter mutant cells expressed reduced levels of the homing molecule CD62L. They also migrated more efficiently towards sphingosine-1-phosphate in vitro and displayed an increased abundance of the sphingosine-1-phosphate receptor 1, compatible with improved lymphocyte egress in vivo. In line with these observations, the ablation of the miR-142 locus in B cells caused a paucity of B cells in the lymph nodes. Mutant B cell accumulation in the latter tissues was also compromised upon transfer into a wild-type environment. These changes coincided with suboptimal levels of FOXO1, a positive regulator of CD62L transcription, in mutant B cells. Overall, our findings indicate contributions for the miR-142 locus in various aspects of the B cell life cycle. Notably, this locus appears to favor the establishment of the migratory behavior required for naïve follicular B cell patrolling activity.

A role for the nuclear receptor NR2F6 in peritoneal B cell homeostasis.

B cells are key mediators of humoral immunity. Mature B cells fall into various sub-classes that can be separated by their ontogeny, expression of cell surface markers, anatomical location, and function. B1 subsets play important roles in natural immunity and constitute the majority of B cells in newborns. In the adult, B1 cells predominate in the pleural and peritoneal cavities, while the mature B2 follicular subset makes up the major fraction of B cells in lymphoid tissue, although important subsets of antibody-secreting B1 cells are also present at these sites. B1 cells are the main producers of natural IgM but can also contribute to elimination of some pathogens, while B2 cells primarily mediate response to foreign antigens. The differential molecular underpinning of the B1 and B2 subsets remains incompletely understood. Here we demonstrate that germline-deficiency of the orphan nuclear receptor NR2F6 causes a partial loss of B1b and B2 B cells in the peritoneum while leaving peritoneal B1a cells unaltered. A competitive bone marrow chimera in Nr2f6+/+ host mice produced similar numbers of Nr2f6+/+ and Nr2f6-/- peritoneal B1b and B2 cells. The proliferation of Nr2f6-/- peritoneal B cells was not altered, while the migration marker CXCR5 was reduced on all subsets but Beta7-integrin was reduced only on peritoneal B1b and B2 cells. Similarly, B1b and B2 but not B1a cells, exhibited significantly reduced survival.

Addressing the role of PKD3 in the T cell compartment with knockout mice.

BACKGROUND: The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far. METHODS: Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology. RESULTS: PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4+ T cells express more interleukin-2 than wild type CD4+ T cells upon TCR stimulation ex vivo. However, purified naïve CD4+ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells. CONCLUSION: While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4+ T cell differentiation in vitro. Video Abstract.

Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ.

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127-KLRG1+) or memory precursors cells (MPECs, CD127+KLRG1-) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8+ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6-/- OT-I T-cells showed that the augmented memory formation is CD8+ T-cell intrinsic. Although the relative difference between the Nr2f6+/+ and Nr2f6-/- OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8+ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8+ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8+ T cells in vivo.

Bacterial Infection with Listeria monocytogenes in Mice and Subsequent Analysis of Antigen-Specific CD8 T Cell Responses.

Pathogens such as bacteria, viruses, fungi, or protozoa can cause acute and chronic infections in their hosts. The intracellular bacterium Listeria monocytogenes serves as a model pathogen to assess the molecular mechanisms regulating CD8 T cell activation, differentiation, and function. We set up an experimental workflow to investigate cell-intrinsic roles of the nuclear receptor NR2F6 in CD8 T cell memory formation upon Listeria monocytogenes (LmOVA) infection ( Jakic et al., 2021 ). The current protocol details how to cultivate ovalbumin-expressing LmOVA, infect naïve C57BL/6 mice with these bacteria and determine the bacterial load in host organs. Furthermore, we describe how to evaluate antigen-specific CD8 T cell responses and discriminate between short-lived effector and memory precursor cells in vivo following LmOVA infection (Figure 1). To assess CD8 T cell-intrinsic molecular mechanisms, we integrated an adoptive cell transfer (ACT) experiment of genetically modified naïve OT-I CD8 T cells into congenic hosts before LmOVA infection. Graphic abstract: Figure 1.Experimental workflow depicting the steps for infection of mice with Listeria and subsequent analysis of antigen-specific CD8 memory responses. Bacteria (ovalbumin expressing Listeria monocytogenes) are thawed and grown on lysogeny broth (LB) plates overnight (ON). A single colony is picked and grown in LB medium ON. Bacteria from the exponential growth phase are then injected into a C57BL/6 mouse via tail vein injection. Colony forming units (CFU) of the bacteria can be detected in the spleen on day 3 post injection. Antigen-specific CD8 T cell immune response can be investigated during the acute phase (d3 after infection), during the peak of the adaptive immune response (d7), the clearance phase (d26), or the memory phase (d70) by flow cytometry. Created with BioRender.com.

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Cell signaling and the aging of B cells.

The uniqueness of each B cell lies in the structural diversity of the B-cell antigen receptor allowing the virtually limitless recognition of antigens, a necessity to protect individuals against a range of challenges. B-cell development and response to stimulation are exquisitely regulated by a group of cell surface receptors modulating various signaling cascades and their associated genetic programs. The effects of these signaling pathways in optimal antibody-mediated immunity or the aberrant promotion of immune pathologies have been intensely researched in the past in young individuals. In contrast, we are only beginning to understand the contribution of these pathways to the changes in B cells of old organisms. Thus, critical transcription factors such as E2A and STAT5 show differential expression or activity between young and old B cells. As a result, B-cell physiology appears altered, and antibody production is impaired. Here, we discuss selected phenotypic changes during B-cell aging and attempt to relate them to alterations of molecular mechanisms.

Context-specific regulation of cell survival by a miRNA-controlled BIM rheostat.

Knockout of the ubiquitously expressed miRNA-17∼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17∼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17∼92:Bim interactions to the complex miR-17∼92 knockout phenotype, we used a system of conditional mutagenesis of the nine Bim 3' UTR miR-17∼92 seed matches. Blocking miR-17∼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17∼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by Bim haploinsufficiency. Thus, the interaction of miR-17∼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.

Orphan Nuclear Receptor NR2F6 Suppresses T Follicular Helper Cell Accumulation through Regulation of IL-21.

CD4 T follicular helper (Tfh) cells are specialized in helping B cells during the germinal center (GC) reaction and ultimately promote long-term humoral immunity. Here we report that loss of the nuclear orphan receptor NR2F6 causes enhanced survival and accumulation of Tfh cells, GC B cells, and plasma cells (PCs) following T cell-dependent immunization. Nr2f6-deficient CD4 T cell dysfunction is the primary cause of cell accumulation. Cytokine expression in Nr2f6-deficient Tfh cells is dysregulated, and Il21 expression is enhanced. Mechanistically, NR2F6 binds directly to the interleukin 21 (IL-21) promoter and a conserved noncoding sequence (CNS) near the Il21 gene in resting CD4+ T cells. During Tfh cell differentiation, this direct NR2F6 DNA interaction is abolished. Enhanced Tfh cell accumulation in Nr2f6-deficient mice can be reverted by blocking IL-21R signaling. Thus, NR2F6 is a critical negative regulator of IL-21 cytokine production in Tfh cells and prevents excessive Tfh cell accumulation.

TET enzymes control antibody production and shape the mutational landscape in germinal centre B cells.

Upon activation by antigen, B cells form germinal centres where they clonally expand and introduce affinity-enhancing mutations into their B-cell receptor genes. Somatic mutagenesis and class switch recombination (CSR) in germinal centre B cells are initiated by the activation-induced cytidine deaminase (AID). Upon germinal centre exit, B cells differentiate into antibody-secreting plasma cells. Germinal centre maintenance and terminal fate choice require transcriptional reprogramming that associates with a substantial reconfiguration of DNA methylation patterns. Here we examine the role of ten-eleven-translocation (TET) proteins, enzymes that facilitate DNA demethylation and promote a permissive chromatin state by oxidizing 5-methylcytosine, in antibody-mediated immunity. Using a conditional gene ablation strategy, we show that TET2 and TET3 guide the transition of germinal centre B cells to antibody-secreting plasma cells. Optimal AID expression requires TET function, and TET2 and TET3 double-deficient germinal centre B cells show defects in CSR. However, TET2/TET3 double-deficiency does not prevent the generation and selection of high-affinity germinal centre B cells. Rather, combined TET2 and TET3 loss-of-function in germinal centre B cells favours C-to-T and G-to-A transition mutagenesis, a finding that may be of significance for understanding the aetiology of B-cell lymphomas evolving in conditions of reduced TET function.

CHK1 dosage in germinal center B cells controls humoral immunity.

Germinal center (GC) B cells are among the fastest replicating cells in our body, dividing every 4-8 h. DNA replication errors are intrinsically toxic to cells. How GC B cells exert control over the DNA damage response while introducing mutations in their antibody genes is poorly understood. Here, we show that the DNA damage response regulator Checkpoint kinase 1 (CHK1) is essential for GC B cell survival. Remarkably, effective antibody-mediated immunity relies on optimal CHK1 dosage. Chemical CHK1 inhibition or loss of one Chk1 allele impairs the survival of class-switched cells and curbs the amplitude of antibody production. Mechanistically, active B cell receptor signaling wires the outcome of CHK1-inhibition towards BIM-dependent apoptosis, whereas T cell help favors temporary cell cycle arrest. Our results predict that therapeutic CHK1 inhibition in cancer patients may prove potent in killing B cell lymphoma and leukemia cells addicted to B cell receptor signaling, but will most likely dampen humoral immunity.

miR-17∼92 in lymphocyte development and lymphomagenesis.

microRNAs (miRNAs) down-modulate the levels of proteins by sequence-specific binding to their respective target mRNAs, causing translational repression or mRNA degradation. The miR-17∼92 cluster encodes for six miRNAs whose target recognition specificities are determined by their distinct sequence. In mice, the four miRNA families generated from the miR-17∼92 cluster coordinate to allow for proper lymphocyte development and effective adaptive immune responses following infection or immunization. Lymphocyte development and homeostasis rely on tight regulation of PI3K signaling to avoid autoimmunity or immunodeficiency, and the miR-17∼92 miRNAs appear as key mediators to appropriately tune PI3K activity. On the other hand, in lymphoid tumors overexpression of the miR-17∼92 miRNAs is a common oncogenic event. In this review, we touch on what we have learned so far about the miR-17∼92 miRNAs, particularly with respect to their role in lymphocyte development, homeostasis and pathology.

The c-Myc/miR17-92/PTEN Axis Tunes PI3K Activity to Control Expression of Recombination Activating Genes in Early B Cell Development.

Appropriate PI3K signals generated by the antigen receptor are essential to promote B cell development. Regulation of recombination activating gene (RAG)-1 and RAG-2 expression is one key process that is mediated by PI3K to ensure developmental progression and selection. When PI3K signals are too high or too low, expression of RAGs does not turn off and B cell development is impaired or blocked. Yet, the mechanism which tunes PI3K activity to control RAG expression during B cell development in the bone marrow is unknown. Recently we showed that a c-Myc/miR17-92/PTEN axis regulates PI3K activity for positive and negative selection of immature B cells. Here, we show that the c-Myc/miR17-92/PTEN axis tunes PI3K activity to control the expression of RAGs in proB cells. Using different genetically engineered mouse models we show that impaired function of the c-Myc/miR17-92/PTEN axis alters the PI3K/Akt/Foxo1 pathway to result in dis-regulated expression of RAG and a block in B cell development. Studies using 38c-13 B lymphoma cells, where RAGs are constitutively expressed, suggest that this regulatory effect is mediated post-translationally through Foxo1.

Differential effects of Vav-promoter-driven overexpression of BCLX and BFL1 on lymphocyte survival and B cell lymphomagenesis.

Overexpression of BCLX and BFL1/A1 has been reported in various human malignancies and is associated with poor prognosis and drug resistance, identifying these prosurvival BCL2 family members as putative drug targets. We have generated transgenic mice that express human BFL1 or human BCLX protein throughout the haematopoietic system under the control of the Vav gene promoter. Haematopoiesis is normal in both the Vav-BFL1 and Vav-BCLX transgenic (TG) mice and susceptibility to spontaneous haematopoietic malignancies is not increased. Lymphoid cells from Vav-BCLX TG mice exhibit increased resistance to apoptosis in vitro while most blood cell types form Vav-BFL1 TG mice were poorly protected. Both transgenes significantly accelerated lymphomagenesis in Eµ-MYC TG mice and, surprisingly, the Vav-BFL1 transgene was the more potent. Unexpectedly, expression of transgenic BFL1 RNA and protein is significantly elevated in B lymphoid cells of Vav-BFL1/Eµ-MYC double-transgenic compared to Vav-BFL1 mice, even during the preleukaemic phase, providing a rationale for the potent synergy. In contrast, Vav-BCLX expression was not notably different. These mouse models of BFL1 and BCLX overexpression in lymphomas should be useful tools for the testing the efficacy of novel human BFL1- and BCLX-specific inhibitors.

Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis.

Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression in response to DNA damage. Despite its essential function, CHK1 has been identified as a target to kill cancer cells and studies using Chk1 haploinsufficient mice initially suggested a role as tumor suppressor. Here, we report on the key role of CHK1 in normal B-cell development, lymphomagenesis and cell survival. Chemical CHK1 inhibition induces BCL2-regulated apoptosis in primary as well as malignant B-cells and CHK1 expression levels control the timing of lymphomagenesis in mice. Moreover, total ablation of Chk1 in B-cells arrests their development at the pro-B cell stage, a block that, surprisingly, cannot be overcome by inhibition of mitochondrial apoptosis, as cell cycle arrest is initiated as an alternative fate to limit the spread of damaged DNA. Our findings define CHK1 as essential in B-cell development and potent target to treat blood cancer.

Transient apoptosis inhibition in donor stem cells improves hematopoietic stem cell transplantation.

During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.

A c-Myc/miR17-92/Pten Axis Controls PI3K-Mediated Positive and Negative Selection in B Cell Development and Reconstitutes CD19 Deficiency.

PI3K activity determines positive and negative selection of B cells, a key process for immune tolerance and B cell maturation. Activation of PI3K is balanced by phosphatase and tensin homolog (Pten), the PI3K's main antagonistic phosphatase. Yet, the extent of feedback regulation between PI3K activity and Pten expression during B cell development is unclear. Here, we show that PI3K control of this process is achieved post-transcriptionally by an axis composed of a transcription factor (c-Myc), a microRNA (miR17-92), and Pten. Enhancing activation of this axis through overexpression of miR17-92 reconstitutes the impaired PI3K activity for positive selection in CD19-deficient B cells and restores most of the B cell developmental impairments that are evident in CD19-deficient mice. Using a genetic approach of deletion and complementation, we show that the c-Myc/miR17-92/Pten axis critically controls PI3K activity and the sensitivity of immature B cells to negative selection imposed by activation-induced cell death.