Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection

Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection. During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst

The MRN complex is transcriptionally regulated by MYCN during neural cell proliferation to control replication stress

The MRE11/RAD50/NBS1 (MRN) complex is a major sensor of DNA double strand breaks, whose role in controlling faithful DNA replication and preventing replication stress is also emerging. Inactivation of the MRN complex invariably leads to developmental and/or degenerative neuronal defects, the pathogenesis of which still remains poorly understood. In particular, NBS1 gene mutations are associated with microcephaly and strongly impaired cerebellar development, both in humans and in the mouse model. These phenotypes strikingly overlap those induced by inactivation of MYCN, an essential promoter of the expansion of neuronal stem and progenitor cells, suggesting that MYCN and the MRN complex might be connected on a unique pathway essential for the safe expansion of neuronal cells. Here, we show that MYCN transcriptionally controls the expression of each component of the MRN complex. By genetic and pharmacological inhibition of the MRN complex in a MYCN overexpression model and in the more physiological context of the Hedgehog-dependent expansion of primary cerebellar granule progenitor cells, we also show that the MRN complex is required for MYCN-dependent proliferation. Indeed, its inhibition resulted in DNA damage, activation of a DNA damage response, and cell death in a MYCN- and replication-dependent manner. Our data indicate the MRN complex is essential to restrain MYCN-induced replication stress during neural cell proliferation and support the hypothesis that replication-born DNA damage is responsible for the neuronal defects associated with MRN dysfunctions.Cell Death and Differentiation advance online publication, 12 June 2015; doi:10.1038/cdd.2015.81

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation

Somatic hypermutation and affinity maturation analysis using the 4-hydroxy-3-nitrophenyl-acetyl (NP) system

Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell-dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to (1) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to (2) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response

Search for New Physics with Jets and Missing Transverse Momentum in pp collisions at sqrt(s) = 7 TeV

A search for new physics is presented based on an event signature of at least three jets accompanied by large missing transverse momentum, using a data sample corresponding to an integrated luminosity of 36 inverse picobarns collected in proton--proton collisions at sqrt(s)=7 TeV with the CMS detector at the LHC. No excess of events is observed above the expected standard model backgrounds, which are all estimated from the data. Exclusion limits are presented for the constrained minimal supersymmetric extension of the standard model. Cross section limits are also presented using simplified models with new particles decaying to an undetected particle and one or two jets

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE

X-ray emission from the Sombrero galaxy: discrete sources

We present a study of discrete X-ray sources in and around the bulge-dominated, massive Sa galaxy, Sombrero (M104), based on new and archival Chandra observations with a total exposure of ~200 ks. With a detection limit of L_X = 1E37 erg/s and a field of view covering a galactocentric radius of ~30 kpc (11.5 arcminute), 383 sources are detected. Cross-correlation with Spitler et al.'s catalogue of Sombrero globular clusters (GCs) identified from HST/ACS observations reveals 41 X-rays sources in GCs, presumably low-mass X-ray binaries (LMXBs). We quantify the differential luminosity functions (LFs) for both the detected GC and field LMXBs, whose power-low indices (~1.1 for the GC-LF and ~1.6 for field-LF) are consistent with previous studies for elliptical galaxies. With precise sky positions of the GCs without a detected X-ray source, we further quantify, through a fluctuation analysis, the GC LF at fainter luminosities down to 1E35 erg/s. The derived index rules out a faint-end slope flatter than 1.1 at a 2 sigma significance, contrary to recent findings in several elliptical galaxies and the bulge of M31. On the other hand, the 2-6 keV unresolved emission places a tight constraint on the field LF, implying a flattened index of ~1.0 below 1E37 erg/s. We also detect 101 sources in the halo of Sombrero. The presence of these sources cannot be interpreted as galactic LMXBs whose spatial distribution empirically follows the starlight. Their number is also higher than the expected number of cosmic AGNs (52+/-11 [1 sigma]) whose surface density is constrained by deep X-ray surveys. We suggest that either the cosmic X-ray background is unusually high in the direction of Sombrero, or a distinct population of X-ray sources is present in the halo of Sombrero.Comment: 11 figures, 5 tables, ApJ in pres