Basal Immunoglobulin Signaling Actively Maintains Developmental Stage in Immature B Cells

In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking “back-differentiation” of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms

NMDA receptors and BAX are essential for Aβ impairment of LTP

Accumulation of amyloid-β (Aβ) is a hallmark of Alzheimer’s disease, a neurodegenerative disorder in which synapse loss and dysfunction are early features. Acute exposure of hippocampal slices to Aβ leads to changes in synaptic plasticity, specifically reduced long-term potentiation (LTP) and enhanced long-term depression (LTD), with no change in basal synaptic transmission. We also report here that D-AP5, a non-selective NMDA receptor antagonist, completely prevented Aβ-mediated inhibition of LTP in area CA1 of the hippocampus. Ro25-6981, an antagonist selective for GluN2B (NR2B) NMDA receptors, only partially prevented this Aβ action, suggesting that GluN2A and GluN2B receptors may both contribute to Aβ suppression of LTP. The effect of Aβ on LTP was also examined in hippocampal slices from BAX −/− mice and wild-type littermates. Aβ failed to block LTP in hippocampal slices from BAX −/− mice, indicating that BAX is essential for Aβ inhibition of LTP

MRI evaluation of dendritic cell vaccination protocol in a murine model of breast cancer (MMTV-hRas)

Introduction: Dendritic cells (DCs) play a pivotal role in the regulation of immune responses and in tumor immunosurveillance. The aim of this study was the set up and the evaluation using MRI of a tumor-specific DC-based vaccine . Methods: Total bone marrow cells were extracted from wt mice; DC differentiation, culture, labelling and antigen loading was performed as previously published (Martelli et al, Mol Imaging Biol 2011). Cell activation was analysed by RealTime PCR. For the vaccination protocol, MNP labelled and antigen loaded DCs were injected weekly into the footpad of transgenic mice (MMTV-hRas) for three weeks (n=4, starting at 7 weeks of age, n=4 control mice) in presence of TNF\u3b1 stimulation. MRI was performed -4, 24 and 48h after cells injection- with a 7T Bruker Pharmascan instrument. Weekly MRI screening were done to identify the presence of pre-neoplastic lesions and the early tumor onset. Notably, MRI analysis also permitted to evaluate the composition of the tumor mass (haemorrhagic, necrotic or vital areas). Plasma was collected at multiple time points. Mice were sacrificed 2 weeks after the tumor onset; and spleen, lymph nodes and tumors were collected for ex vivo analysis. Splenocytes were re-exposed to antigen loaded DCs, to evaluate the presence of a specific immune response against tumor antigens. Paraffin embedded tumors were used for immunohistochemical analysis. Results: Gene expression profile showed the up-regulation of multiple genes involved in the processing (e.g. B2m) and presentation (e.g. CD36 and CD40) of antigens, and in the production of cytokines (e.g. IL-12, CXCL10 and CXCL12). MRI showed lymph nodal homing of the iron labelled activated DCs cells injected into the footpad of MMTV-hRas mice; these data were confirmed by immunohistochemistry, showing co-localization of CD208 positive cells (but not macrophages) and iron. MRI screening of mammary glands showed a delay in tumor onset in vaccinated mice compared to control ones (4 weeks). Cytokine quantification in plasma samples showed a statistically significant increase of IL-12 production in vaccinated mice, compared with controls, at tumor onset. Cytofluorimetric analysis of immune cells derived from spleen demonstrated a systemic activation, characterized by an augmented production of perforin by CD8 and NK cells and of activatory cytokines (e.g. IL-2, TNF\u3b1 e IFN\u3b3) by CD4 and CD8 cells after re-exposition to antigen. Immunohistochemical analysis suggested a shift in the M1/M2 tumor associated macrophages ratio towards the anti-tumoral ones (M1>M2) in vaccinated mice. Conclusions: Dynamic in vivo MRI monitoring during the vaccine protocol provides insight for the early evaluation of a vaccine potential (DC migration to LNs) and for the assessment of tumor onset time. This non-invasive approach offers a powerful and highly translational tool for the set up and optimization of cell mediated treatment in clinical trials

Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk

The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy

Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway