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Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes and neurological disorders (1). Genetic manipulation in the rat is hampered by the lack of suitable technologies such as embryonic stem cells (ES) which are routinely used to generate targeted mutations in the mouse. Cloning through somatic cell nuclear transfer (SCNT) is a potential alternative approach in species for which ES technologies are unavailable. However, all previous efforts to clone rats have been unsuccessful, with developmental arrest at implantation stage (2 and references therein). The fine-tuned coordination between nuclear transfer and timing of oocyte activation is critical to the outcome of somatic cloning. This coordination is hampered in the rat because almost all the oocytes spontaneously, although abortively, activate within 60 min of their removal from oviducts (3). Such rapid but incomplete activation process is not encountered in other cloned species. To allow embryo reconstruction before the onset of oocyte activation, we initially developed a one-step SCNT procedure for the rapid substitution of the endogenous meiotic metaphase by an exogenous mitotic one. This latter was isolated from synchronized cultured fetal CD-SpragueDawley fibroblasts (12.5 dpc). Individual mitotic nuclei were injected into a recipient OFA-SpragueDawley oocyte, from which the meiotic metaphase was withdrawn while removing the micropipette from cytoplasm after injection. However, within 30 min after recovery 70% of oocytes showed clear morphological evidence of spontaneous release from MII (Fig. 1A). When activation of cloned embryos (Fig. 1B) was induced and maintained by exposure to a cdc2-specific kinase inhibitor (Butyrolactone;150 µM, 2 h) (4), 201 out of 221 reconstructed embryos expelled the polar body and subsequently divided into two-cell embryos. Their transfer into OFA-SpragueDawley foster mothers (11 recipients, 221 embryos) resulted in 9 implantation sites, but no fetal development. Forty percent of oocytes selected for SCNT turned to be already activated as evidenced by disjoined sister chromatids moving to opposite poles (Fig. 1C). These observations strongly supported the view that despite rapid manipulation, most of the oocytes were not suitable for cloning. As activation is triggered by the inactivation of MPF activity through a proteasome-mediated cyclin degradation pathway, we used MG132, a protease inhibitor that reversibly blocks the first meiotic metaphase-anaphase transition in the rat (5). We found that this drug also reversibly stabilized most oocyte MII metaphases for up to 3 hours (77%; Fig. 1D). We then collected oocytes in the presence of MG132 (5µM) (4) as described above. SCNT was performed within 30 minutes of drug removal. 876 embryos were implanted into 12 pseudopregnant foster female rats. At 12.5 dpc, the females were sacrificed, and four females contained 16 fetuses. Thirteen of the fetuses, obtained from 3 females, were viable with beating hearts (Fig 1E). In the next series of experiments, we transplanted 129 cloned embryos into two foster mothers and allowed them to go to term. Only one foster mother contained viable fetuses and this animal delivered three live male pups of fibroblast origin as unambiguously demonstrated by microsatellite marker analysis (4). One normal sized pup (5.9 g) died a few hours after birth. The other two pups grew to sexual maturity (Fig. 1F) and generated normal progeny. We have also obtained normal progenies (size, weight and development) from two additional cloned female rats demonstrating the potential of the technique for the development of fertile rat lines of both sexes (supporting online text). Our data highlight the importance of adapting the SCNT procedure to oocyte physiology for successful cloning. Recently, random mutagenesis has been proposed to generate knockout rats (6). Our results however pave the way for more extensive genetic modifications such as conditional knockout and gene replacement which are required to produce relevant models of human diseases.

Recent studies have shown that complement activation is not confined to the serum but also occurs within cellular compartments. This has led to an emerging understanding that complement components can intersect diverse cellular metabolic and effector pathways. Here, the authors propose that the different locations of complement activation dictate its diverse functions. Complement is traditionally known to be a system of serum proteins that provide protection against pathogens through direct cell lysis and the mobilization of innate and adaptive immunity. However, recent work indicates that the complement system has additional physiological roles beyond those in host defence. In this Opinion article, we describe the new modes and locations of complement activation that enable it to interact with other cell effector systems, such as growth factor receptors, inflammasomes and metabolic pathways. We propose that the location of complement activation dictates its function.

The complement receptor CD46 and the Notch-Jagged system are important for the differentiation of helper T cells. Kemper and colleagues demonstrate that Jagged1 is a physiological ligand for CD46 and is critical for the generation of T helper type 1 cells in humans. CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (T H 1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4 + T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate T H 1 responses in vitro and in vivo , which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients.

The induction of human CD4 + Th1 cells requires autocrine stimulation of the complement receptor CD46 in direct crosstalk with a CD4 + T cell-intrinsic NLRP3 inflammasome. However, it is unclear whether human cytotoxic CD8 + T cell (CTL) responses also rely on an intrinsic complement-inflammasome axis. Here we show, using CTLs from patients with CD46 deficiency or with constitutively-active NLRP3, that CD46 delivers co-stimulatory signals for optimal CTL activity by augmenting nutrient-influx and fatty acid synthesis. Surprisingly, although CTLs express NLRP3, a canonical NLRP3 inflammasome is not required for normal human CTL activity, as CTLs from patients with hyperactive NLRP3 activity function normally. These findings establish autocrine complement and CD46 activity as integral components of normal human CTL biology, and, since CD46 is only present in humans, emphasize the divergent roles of innate immune sensors between mice and men. Complement, while serving to remove pathogens in the circulation, is also important for synergizing with inflammasomes to modulate CD4 T cell activation. Here the authors show that CD46, a complement receptor expressed only in humans, is essential for inducing optimal activation and effector functions of human CD8 T cells.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1 ). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins 2 – 9 . NL63 uses angiotensin-converting enzyme 2 as a receptor 10 , whereas 229E uses human aminopeptidase-N 11 . HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown 12 . Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell–cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry. We demonstrate that the transmembrane protease TMPRSS2 is a receptor for coronavirus HKU1; it triggers HKU1-mediated cell–cell fusion and viral entry by binding to both HKU1A and HKU1B spikes.

The complement system has long been known to be a major element of innate immunity. Traditionally, it was regarded as the first line of defense against invading pathogens, leading to opsonization and phagocytosis or the direct lysis of microbes. However, from the second half of the twentieth century on, it became clear that complement is also intimately involved in the induction and “fine tuning” of adaptive B- and T-cell responses as well as lineage commitment. This growing recognition of the complement system's multifunctional role in immunity is consistent with the recent paradigm that complement is also necessary for the successful contraction of an adaptive immune response. This review aims at giving a condensed overview of complement's rise from a simple innate stop-and-go system to an essential and efficient participant in general immune homeostasis and acquired immunity.

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.

CD46 is best known as a regulator of complement function. Kemper et al . show that CD46 can switch inflammatory T cells into a regulatory mode and demonstrate the molecular details of this process. In this study we demonstrate a new form of immunoregulation: engagement on CD4 + T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (T H 1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4 + T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4 + T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-γ (IFN-γ). Finally, γδ T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) γδ and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.

Signaling via the complement factors C3a and C5a regulates effector T cell responses. Evidence now links the absence of local complement activation with a default pathway that leads to the polarization of Foxp3 + regulatory T cells.