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.

Pretargeted Imaging with Gallium-68-Improving the Binding Capability by Increasing the Number of Tetrazine Motifs.

The inverse electron-demand Diels-Alder reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooct-2-ene (TCO) has gained increasing attraction among extensive studies on click chemistry due to its exceptionally fast reaction kinetics and high selectivity for in vivo pretargeting applications including PET imaging. The facile two-step approach utilizing TCO-modified antibodies as targeting structures has not made it into clinics yet. An increase in the blood volume of humans in comparison to mice seems to be the major limitation. This study aims to show if the design of multimeric Tz-ligands by chelator scaffolding can improve the binding capacity and may lead to enhanced PET imaging with gallium-68. We utilized for this purpose the macrocyclic siderophore Fusarinine C (FSC) which allows conjugation of up to three Tz-residues due to three primary amines available for site specific modification. The resulting mono- di- and trimeric conjugates were radiolabelled with gallium-68 and characterized in vitro (logD, protein binding, stability, binding towards TCO modified rituximab (RTX)) and in vivo (biodistribution- and imaging studies in normal BALB/c mice using a simplified RTX-TCO tumour surrogate). The 68Ga-labelled FSC-based Tz-ligands showed suitable hydrophilicity, high stability and high targeting specificity. The binding capacity to RTX-TCO was increased according to the grade of multimerization. Corresponding in vivo studies showed a multimerization typical profile but generally suitable pharmacokinetics with low accumulation in non-targeted tissue. Imaging studies in RTX-TCO tumour surrogate bearing BALB/c mice confirmed this trend and revealed improved targeting by multimerization as increased accumulation in RTX-TCO positive tissue was observed.

DNA-binding of the Tet-transactivator curtails antigen-induced lymphocyte activation in mice.

The Tet-On/Off system for conditional transgene expression constitutes state-of-the-art technology to study gene function by facilitating inducible expression in a timed and reversible manner. Several studies documented the suitability and versatility of this system to trace lymphocyte fate and to conditionally express oncogenes or silence tumour suppressor genes in vivo. Here, we show that expression of the tetracycline/doxycycline-controlled Tet-transactivator, while tolerated well during development and in immunologically unchallenged animals, impairs the expansion of antigen-stimulated T and B cells and thereby curtails adaptive immune responses in vivo. Transactivator-mediated cytotoxicity depends on DNA binding, but can be overcome by BCL2 overexpression, suggesting that apoptosis induction upon lymphocyte activation limits cellular and humoral immune responses. Our findings suggest a possible system-intrinsic biological bias of the Tet-On/Off system in vivo that will favour the outgrowth of apoptosis resistant clones, thus possibly confounding data published using such systems.