Dinis Pedro Calado

Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes

The genomic region encoding the miR-17-92 microRNA (miRNA) cluster is often amplified in lymphoma and other cancers, and cancer cells carrying this amplification have higher expression of miRNA in this cluster. Retroviral expression of miR-17-92 accelerates c-Myc-induced lymphoma development, but precisely how higher expression of miR-17-92 promotes lymphomagenesis remains unclear. Here we generated mice with higher expression of miR-17-92 in lymphocytes. These mice developed lymphoproliferative disease and autoimmunity and died prematurely. Lymphocytes from these mice showed more proliferation and less activation-induced cell death. The miR-17-92 miRNA suppressed expression of the tumor suppressor PTEN and the proapoptotic protein Bim. This mechanism probably contributed to the lymphoproliferative disease and autoimmunity of miR-17-92-transgenic mice and contributes to lymphoma development in patients with amplifications of the miR-17-92 coding region.

MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb.

MiR-150 is a microRNA (miRNA) specifically expressed in mature lymphocytes, but not their progenitors. A top predicted target of miR-150 is c-Myb, a transcription factor controlling multiple steps of lymphocyte development. Combining loss- and gain-of-function gene targeting approaches for miR-150 with conditional and partial ablation of c-Myb, we show that miR-150 indeed controls c-Myb expression in vivo in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations and that this dramatically affects lymphocyte development and response. Our results identify a key transcription factor as a critical target of a stage-specifically expressed miRNA in lymphocytes and suggest that this and perhaps other miRNAs have evolved to control the expression of just a few critical target proteins in particular cellular contexts.

Foxp3-Dependent MicroRNA155 Confers Competitive Fitness to Regulatory T Cells by Targeting SOCS1 Protein

Foxp3(+) regulatory T (Treg) cells limit pathogenic immune responses to self-antigens and foreign antigens. An essential role for microRNA (miRNA) in the maintenance and function of Treg cells, revealed by the Treg cell-specific Dicer ablation, raised a question as to a specific miRNA contribution. We found that Foxp3 controlled the elevated miR155 expression required for maintaining Treg cell proliferative activity and numbers under nonlymphopenic conditions. Moreover, miR155 deficiency in Treg cells resulted in increased suppressor of cytokine signaling 1 (SOCS1) expression accompanied by impaired activation of signal transducer and activator of transcription 5 (STAT5) transcription factor in response to limiting amounts of interleukin-2. Our studies suggest that Foxp3-dependent regulation of miR155 maintains competitive fitness of Treg cell subsets by targeting SOCS1, and they provide experimental support for a proposed role for miRNAs in ensuring the robustness of cellular phenotypes.

PI3 kinase signals BCR-dependent mature B cell survival.

Previous work has shown that mature B cells depend upon survival signals delivered to the cells by their antigen receptor (BCR). To identify the molecular nature of this survival signal, we have developed a genetic approach in which ablation of the BCR is combined with the activation of specific, BCR dependent signaling cascades in mature B cells in vivo. Using this system, we provide evidence that the survival of BCR deficient mature B cells can be rescued by a single signaling pathway downstream of the BCR, namely PI3K signaling, with the FOXO1 transcription factor playing a central role.

Halofuginone Inhibits TH17 Cell Differentiation by Activating the Amino Acid Starvation Response

Starving T Cells The TH17 lineage of CD4+ helper T cells, characterized by the ability to secrete IL-17, is an important mediator of inflammation and autoimmunity. Dampening the responses of these cells or inhibiting their differentiation is of great therapeutic interest. Sundrud et al. (p. 1334; see the Perspective by Blander and Amsen) now show that the small molecule halofuginone inhibits the differentiation of TH17 cells but not other CD4+ T cell helper lineages both in vitro and in a mouse model of multiple sclerosis. This selective inhibition was mediated by activation of the amino acid starvation response. Amino acid depletion mimicked the effects of halofuginone, whereas excess amino acids rescued TH17 differentiation. The results highlight the importance of amino acid metabolism in regulating inflammation. Activation of the amino acid starvation response inhibits differentiation of a subset of inflammatory T cells. A central challenge for improving autoimmune therapy is preventing inflammatory pathology without inducing generalized immunosuppression. T helper 17 (TH17) cells, characterized by their production of interleukin-17, have emerged as important and broad mediators of autoimmunity. Here we show that the small molecule halofuginone (HF) selectively inhibits mouse and human TH17 differentiation by activating a cytoprotective signaling pathway, the amino acid starvation response (AAR). Inhibition of TH17 differentiation by HF is rescued by the addition of excess amino acids and is mimicked by AAR activation after selective amino acid depletion. HF also induces the AAR in vivo and protects mice from TH17-associated experimental autoimmune encephalomyelitis. These results indicate that the AAR pathway is a potent and selective regulator of inflammatory T cell differentiation in vivo.

Synergy between PI3K Signaling and MYC in Burkitt Lymphomagenesis

In Burkitt lymphoma (BL), a germinal center B-cell-derived tumor, the pro-apoptotic properties of c-MYC must be counterbalanced. Predicting that survival signals would be delivered by phosphoinositide-3-kinase (PI3K), a major survival determinant in mature B cells, we indeed found that combining constitutive c-MYC expression and PI3K activity in germinal center B cells of the mouse led to BL-like tumors, which fully phenocopy human BL with regard to histology, surface and other markers, and gene expression profile. The tumors also accumulate tertiary mutational events, some of which are recurrent in the human disease. These results and our finding of recurrent PI3K pathway activation in human BL indicate that deregulated c-MYC and PI3K activity cooperate in BL pathogenesis.

The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers

Germinal centers (GCs) are sites of intense B cell proliferation and are central for T cell–dependent antibody responses. However, the role of c-Myc, a key cell-cycle regulator, in this process has been questioned. Here we identified c-Myc+ B cell subpopulations in immature and mature GCs and found, by genetic ablation of Myc, that they had indispensable roles in the formation and maintenance of GCs. The identification of these functionally critical cellular subsets has implications for human B cell lymphomagenesis, which originates mostly from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the c-Myc+ GC subpopulations may be at a particularly high risk for malignant transformation.

Constitutive Canonical NF-κB Activation Cooperates with Disruption of BLIMP1 in the Pathogenesis of Activated B Cell-like Diffuse Large Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) comprises disease entities with distinct genetic profiles, including germinal center B cell (GCB)-like and activated B cell (ABC)-like DLBCLs. Major differences between these two subtypes include genetic aberrations leading to constitutive NF-κB activation and interference with terminal B cell differentiation through BLIMP1 inactivation, observed in ABC- but not GCB-DLBCL. Using conditional gain-of-function and/or loss-of-function mutagenesis in the mouse, we show that constitutive activation of the canonical NF-κB pathway cooperates with disruption of BLIMP1 in the development of a lymphoma that resembles human ABC-DLBCL. Our work suggests that both NF-κB signaling, as an oncogenic event, and BLIMP1, as a tumor suppressor, play causal roles in the pathogenesis of ABC-DLBCL.

NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells

BAFF-R-dependent activation of the alternative NF-κB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-κB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal p100 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-κB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked.

Immature B cells preferentially switch to IgE with increased direct Sμ to Sε recombination

To be added.