Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.

The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages.

Essential Function for the Nuclear Protein Akirin2 in B Cell Activation and Humoral Immune Responses.

Akirin2, an evolutionarily conserved nuclear protein, is an important factor regulating inflammatory gene transcription in mammalian innate immune cells by bridging the NF-κB and SWI/SNF complexes. Although Akirin is critical for Drosophila immune responses, which totally rely on innate immunity, the mammalian NF-κB system is critical not only for the innate but also for the acquired immune system. Therefore, we investigated the role of mouse Akirin2 in acquired immune cells by ablating Akirin2 function in B lymphocytes. B cell-specific Akirin2-deficient (Cd19(Cre/+)Akirin2(fl/fl)) mice showed profound decrease in the splenic follicular (FO) and peritoneal B-1, but not splenic marginal zone (MZ), B cell numbers. However, both Akirin2-deficient FO and MZ B cells showed severe proliferation defect and are prone to undergo apoptosis in response to TLR ligands, CD40, and BCR stimulation. Furthermore, B cell cycling was defective in the absence of Akirin2 owing to impaired expression of genes encoding cyclin D and c-Myc. Additionally, Brg1 recruitment to the Myc and Ccnd2 promoter was severely impaired in Akirin2-deficient B cells. Cd19(Cre/+)Akirin2(fl/fl) mice showed impaired in vivo immune responses to T-dependent and -independent Ags. Collectively, these results demonstrate that Akirin2 is critical for the mitogen-induced B cell cycle progression and humoral immune responses by controlling the SWI/SNF complex, further emphasizing the significant function of Akirin2 not only in the innate, but also in adaptive immune cells.

Cyclin D3 is selectively required for proliferative expansion of germinal center B cells.

The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.

CD70-CD27 interactions provide survival and proliferative signals that regulate T cell receptor-driven activation of human γδ peripheral blood lymphocytes.

Human Vγ9Vδ2 T cells are potent anti-tumor lymphocytes that specifically respond to pyrophosphate (phospho-) antigens, which constitute the basis of current γδ T-cell-based immunotherapy strategies. Despite a clear involvement of the TCR, the costimulation requirements of Vγ9Vδ2 T cells remain ill-defined. Here, we show that the expression of the CD27 receptor by the vast majority of Vγ9Vδ2 peripheral blood lymphocytes endows them with enhanced proliferative capacity upon ligation by its unique ligand CD70, a tumor necrosis factor superfamily member expressed on lymphoma B-cells but also on TCR-activated γδ T cells. Moreover, Vγ9Vδ2 T-cell treatment with soluble recombinant CD70 induced calcium signals and increased transcription of anti-apoptotic Bcl2a1 and cell-cycle-promoting Cyclin D2 genes. We further demonstrate that the manipulation of CD70-CD27 interactions significantly impacted on Vγ9Vδ2 T-cell survival, proliferation and cytokine secretion, in both loss-of-function and gain-of-function experiments. Thus, CD27 coreceptor signals strongly promoted the expansion of Th1-biased, CD27(+) Vγ9Vδ2 peripheral blood lymphocytes in the context of TCR-mediated stimulation with phosphoantigens. These data collectively establish a novel role for the CD70-CD27 axis in human γδ T-cell activation and hence open new perspectives for its modulation in clinical settings.