Sébastien Jaeger

Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46

Natural killer (NK) cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. However, our understanding of NK cell biology is severely limited by the lack of consensus phenotypic definition of these cells across species, by the lack of specific marker to visualize them in situ, and by the lack of a genetic model where NK cells may be selectively ablated. NKp46/CD335 is an Ig-like superfamily cell surface receptor involved in human NK cell activation. In addition to human, we show here that NKp46 is expressed by NK cells in all mouse strains analyzed, as well as in three common monkey species, prompting a unifying phenotypic definition of NK cells across species based on NKp46 cell surface expression. Mouse NKp46 triggers NK cell effector function and allows the detection of NK cells in situ. NKp46 expression parallels cell engagement into NK differentiation programs because it is detected on all NK cells from the immature CD122+NK1.1+DX5− stage and on a minute fraction of NK-like T cells, but not on CD1d-restricted NKT cells. Moreover, human NKp46 promoter drives NK cell selective expression both in vitro and in vivo. Using NKp46 promoter, we generated transgenic mice expressing EGFP and the diphtheria toxin (DT) receptor in NK cells. DT injection in these mice leads to a complete and selective NK cell ablation. This model paves a way for the in vivo characterization and preclinical assessment of NK cell biological function.

Immunoreceptor tyrosine‐based inhibition motifs: a quest in the past and future

Summary: Since an immunoreceptor tyrosine‐based inhibition motif (ITIM) was first identified in the intracytoplasmic domain of FcγRIIB, ITIMs have been found in a large number of inhibitory molecules that were shown to negatively regulate cell activation. Due to their wide tissue distribution and to the variety of their extracellular ligands, ITIM‐containing molecules are involved in the control of a large spectrum of biological functions, mostly but not exclusively related to immunity. On the basis of sequence comparison, ITIMs were structurally defined as 6‐amino acid sequences containing a tyrosine (Y) with loosely conserved N‐terminal (Y−2) and C‐terminal (Y+3) residues. Molecular analysis of signaling events demonstrated that when coaggregated with activating receptors, ITIMs are phosphorylated by Src‐family tyrosine kinases, which enables them to recruit Src homology 2 domain‐containing phosphatases that antagonize activation signals. Because ITIM‐dependent negative regulation seems to be a fundamental regulatory mechanism, both in rodents and in humans, and because it can be used either as a target or as a powerful tool in various diseases, we undertook (i) a genome‐wide search of potential novel ITIM‐containing molecules in humans, mice, frogs, birds, and flies and (ii) a comparative analysis of potential ITIMs in major animal phyla, from mammals to protozoa. We found a surprisingly high number of potential ITIM‐containing molecules, having a great diversity of extracellular domains, and being expressed by a variety of immune and non‐immune cells. ITIMs could be traced back to the most primitive metazoa. The genes that encode ITIM‐containing molecules that belong to the immunoglobulin superfamily or to the C‐lectin family seem to derive from a common set of ancestor genes and to have dramatically expanded and diverged in Gnathostomata (from fish to mammals).

The speed of change: towards a discontinuity theory of immunity?

Immunology — though deeply experimental in everyday practice — is also a theoretical discipline. Recent advances in the understanding of innate immunity, how it is triggered and how it shares features that have previously been uniquely ascribed to the adaptive immune system, can contribute to the refinement of the theoretical framework of immunology. In particular, natural killer cells and macrophages are activated by transient modifications, but adapt to long-lasting modifications that occur in the surrounding tissue environment. This process facilitates the maintenance of self-tolerance while permitting efficient immune responses. In this Essay we extend this idea to other components of the immune system and we propose some general principles that lay the foundations for a unifying theory of immunity — the discontinuity theory. According to this theoretical framework, effector immune responses (namely, activated responses that lead to the potential elimination of the target antigen) are induced by an antigenic discontinuity; that is, by the sudden modification of molecular motifs with which immune cells interact.

CoCAS: A ChIP-on-chip Analysis Suite

Motivation: High-density tiling microarrays are increasingly used in combination with ChIP assays to study transcriptional regulation. To ease the analysis of the large amounts of data generated by this approach, we have developed ChIP-on-chip Analysis Suite (CoCAS), a standalone software suite which implements optimized ChIP-on-chip data normalization, improved peak detection, as well as quality control reports. Our software allows dye swap, replicate correlation and connects easily with genome browsers and other peak detection algorithms. CoCAS can readily be used on the latest generation of Agilent high-density arrays. Also, the implemented peak detection methods are suitable for other datasets, including ChIP-Seq output. Availability: The software is available for download along with a sample dataset at http://www.ciml.univ-mrs.fr/software/ferrier.htm. Contact: ferrier@ciml.univ-mrs.fr; andrau@ciml.univ-mrs.fr; spicuglia@ciml.univ-mrs.fr Supplementary information: Supplementary data are available at Bioinformatics online.

Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro methylated genomic DNA

BackgroundDNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP) assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of in vitro methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples.FindingsWe performed MeDIP assays using in vitro methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using in vitro methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.ConclusionWe illustrate the use of in vitro methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.

Initiation of V(D)J Recombination by Dβ-Associated Recombination Signal Sequences: A Critical Control Point in TCRβ Gene Assembly

T cell receptor (TCR) β gene assembly by V(D)J recombination proceeds via successive Dβ-to-Jβ and Vβ-to-DJβ rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vβ-to-Jβ rearrangements. However the B12/23 restriction does not explain the order of TCRβ assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRβ locus and found that nicks were only detectable at Dβ-associated RSSs. This pattern implies that Dβ 12RSS and, unexpectedly, Dβ 23RSS initiate V(D)J recombination and capture their respective Vβ or Jβ RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dβ1 23RSS impedes cleavage at the adjacent Dβ1 12RSS and consequently Vβ-to-Dβ1 rearrangement first requires the Dβ1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a ‘Dβ1 23RSS-mediated’ restriction operating beyond chromatin accessibility, which directs Dβ1 ordered rearrangements.

Duality of Enhancer Functioning Mode Revealed in a Reduced TCRβ Gene Enhancer Knockin Mouse Model

The TCRβ gene enhancer (Eβ) commands TCRβ gene expression through the lifespan of T lymphocytes. Genetic and molecular studies have implied that in early thymocytes, Eβ directs chromatin opening over the Dβ-Jβ-Cβ domains and triggers initial Dβ-Jβ recombination. In mature T cells, Eβ is required for expression of the assembled TCRβ gene. Whether these separate activities rely on distinct Eβ regulatory sequences and involve differing modes of activation is unclear. Using gene targeting in mouse embryonic stem cells, we replaced Eβ by a conserved core fragment (Eβ169). We found that Eβ169-carrying alleles were capable of sustaining β gene expression and the development of mature T cells in homozygous knockin mice. Surprisingly, these procedures and underlying molecular transactions were affected to a wide range of degrees depending on the developmental stage. Early thymocytes barely achieved Dβ-Jβ germline transcription and recombination. In contrast, T cells displayed substantial though heterogeneous levels of VDJ-rearranged TCRβ gene expression. Our results have implications regarding enhancer function in cells of the adaptive immune system and, potentially, TCRβ gene recombination and allelic exclusion.

Protein synthesis inhibition and GADD34 control IFN‐β heterogeneous expression in response to dsRNA

In innate immune responses, induction of type‐I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type‐I IFN production is linked to cells ability to enter dsRNA‐activated PKR‐dependent translational arrest and then overcome this inhibition by decreasing eIF2α phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti‐viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN‐β mRNA transcription, while GADD34‐dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA‐activated cells.

Experimental detection of long-distance interactions between biomolecules through their diffusion behavior: numerical study.

The dynamical properties and diffusive behavior of a collection of mutually interacting particles are numerically investigated for two types of long-range interparticle interactions: Coulomb-electrostatic and dipole-electrodynamic. It is shown that when the particles are uniformly distributed throughout the accessible space, the self-diffusion coefficient is always lowered by the considered interparticle interactions, irrespective of their attractive or repulsive character. This fact is also confirmed by a simple model to compute the correction to the Brownian diffusion coefficient due to the interactions among the particles. These interactions are also responsible for the onset of dynamical chaos and an associated chaotic diffusion which still follows an Einstein-Fick-like law for the mean-square displacement as a function of time. Transitional phenomena are observed for Coulomb-electrostatic (repulsive) and dipole-electrodynamic (attractive) interactions considered both separately and in competition. The outcomes reported in this paper clearly indicate a feasible experimental method to probe the activation of resonant electrodynamic interactions among biomolecules.

TCR beta allelic exclusion in dynamical models of V(D)J recombination based on allele independence.

Allelic exclusion represents a major aspect of TCRβ gene assembly by V(D)J recombination in developing T lymphocytes. Despite recent progress, its comprehension remains problematic when confronted with experimental data. Existing models fall short in terms of incorporating into a unique distribution all the cell subsets emerging from the TCRβ assembly process. To revise this issue, we propose dynamical, continuous-time Markov chain-based modeling whereby essential steps in the biological procedure (D-J and V-DJ rearrangements and feedback inhibition) evolve independently on the two TCRβ alleles in every single cell while displaying random modes of initiation and duration. By selecting parameters via fitting procedures, we demonstrate the capacity of the model to offer accurate fractions of all distinct TCRβ genotypes observed in studies using developing and mature T cells from wild-type or mutant mice. Selected parameters in turn afford relative duration for each given step, hence updating TCRβ recombination distinctive timings. Overall, our dynamical modeling integrating allele independence and noise in recombination and feedback-inhibition events illustrates how the combination of these ingredients alone may enforce allelic exclusion at the TCRβ locus.